Effect Of Cyclic Adenosine Research Articles

Cre transgene results in global attenuation of the cAMP/PKA pathway

Use of the cre transgene in in vivo mouse models to delete a specific ‘floxed' allele is a well-accepted method for studying the effects of spatially or temporarily regulated genes. During the course of our investigation into the effect of cyclic adenosine 3′,5′-monophosphate-dependent protein kinase A (PKA) expression on cell death, we found that cre expression either in cultured cell lines or in transgenic mice results in global changes in PKA target phosphorylation. This consequently alters gene expression profile and changes in cytokine secretion such as IL-6. These effects are dependent on its recombinase activity and can be attributed to the upregulation of specific inhibitors of PKA (PKI). These results may explain the cytotoxicity often associated with cre expression in many transgenic animals and may also explain many of the phenotypes observed in the context of Cre-mediated gene deletion. Our results may therefore influence the interpretation of data generated using the conventional cre transgenic system.

Effect of Cyclic Adenosine 3′,5′-Monophosphate/Protein Kinase A Pathway on Markers of Cell Proliferation in Nonfunctioning Pituitary Adenomas

Alterations in cAMP signaling have been identified as a cause of endocrine neoplasia. In particular, activating mutations of the G(s)alpha gene and protein kinase A (PKA) overactivity due to low expression of PKA regulatory subunit 1A (R1A) have been implicated in somatotroph proliferation. The objective of this study was to evaluate the effects of cAMP-PKA cascade activation in nonfunctioning pituitary adenomas (NFPA). By immunohistochemistry, R1A, R2A, and R2B expression was evaluated in cells obtained from eight surgically removed NFPA positive for gonadotropins. Cyclin D1 expression and ERK1/2 activity were analyzed under basal conditions and after cAMP-PKA cascade activation. Immunohistochemistry studies demonstrated a low R1/R2 ratio in all NFPA. Additional unbalance of R1/R2 ratio by 8-chloroadenosine cAMP (8-Cl-cAMP) and direct adenylyl cyclase stimulation by forskolin did not increase cyclin D1 expression or ERK1/2 activity in five NFPA (group 1), but even caused 74 +/- 15% and 85 +/- 13% inhibitions of cyclin D1 and ERK1/2 activity, respectively, in the remaining NFPA (group 2). Moreover, in group 2, PKA blockade by the specific inhibitor PKI increased cyclin D1 expression (96 +/- 25% over basal) and ERK1/2 activity (116 +/- 28% over basal). These data show that in contrast with what was previously observed in transformed somatotrophs, activation of the cAMP-PKA pathway did not generate proliferative signals in tumoral cells of the gonadotroph lineage, and in a subset of tumors even exerted a tonic inhibitory effect, thus confirming a different role for the cAMP-mediated pathway in promoting proliferation in the pituitary.

Releasing the block

The inhibitory effect of cyclic adenosine 3',5'-monophosphate (cAMP) on lymphocyte activation is mediated through cAMP-dependent protein kinase (protein kinase A/PKA). Lymphocyte activation is also negatively regulated by a subgroup of protein tyrosine phosphatases (PTPases) that inactivate MAP kinases. The hematopoietic PTPase, HePTP, is known to bind to and rapidly deactivate the MAP kinases Erk1, Erk2, and p38. Hence, HePTP reduces transcription activation from Erk/p38-dependent promoters. Saxena et al. now report that HePTP is phosphorylated by PKA. This phosphorylation event reduced HePTP interaction with its bound MAP kinase. Release from this inhibition resulted in MAP kinase activation and transcription from the c-fos promoter. Hence, crosstalk between the cAMP/PKA pathway and MAP kinase activation pathways may occur through PTPases in immune cells. Saxena, M., Williams, S., Tasken, K., Mustelin, T. (1999) Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase. Nature Cell Biol . 1 : 305-310. Online Journal

Suppression of drug-induced epileptiform discharges by cyclic AMP in rat hippocampus.

The effect of cyclic adenosine 3',5'-monophosphate (cAMP) on epileptiform activity in rat hippocampal slices was investigated. Bath-applied cAMP reversibly decreased the frequency of extracellularly recorded discharges in the CA3 subfield induced by bethanechol- or theophylline-containing solutions. Because cAMP was presumed to be relatively membrane impermeant, we developed and tested the hypothesis that this cAMP-mediated effect occurred extracellularly through the catabolic conversion of cAMP to 5'-AMP and, in turn, to adenosine, a known inhibitory neuromodulator. Three predictions derived from this catabolic hypothesis were tested. First, blockers of the enzymes involved were predicted to antagonize this effect of cAMP. In contrast, the coapplication of a cAMP-phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or a 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate (AMP-CP), enhanced the cAMP-induced suppressive effect. Second, the nonhydrolyzable cAMP analogs, dibutyryl- and 8-bromo-cAMP, were predicted to be ineffective. Low concentrations (5-40 microM) of these two derivatives, however, also suppressed bethanechol-induced discharges, while, at a higher concentration (100 microM), both analogs increased discharge frequencies. Third, enzymatic catabolism of adenosine was predicted to antagonize cAMP's effect, but coapplying adenosine deaminase (10 U/mL) did not diminish this action. Because these data did not support the catabolic hypothesis, other, as yet undefined, mechanisms must be responsible for the discharge-suppressant effect of cAMP.

Cyclic-AMP-stimulated synthesis and release of carcinoembryonic antigen by pancreatic cancer cells.

The effect of cyclic adenosine 3':5'-monophosphate (cAMP) upon the synthesis and release of carcinoembryonic antigen (CEA) was studied in the human pancreatic ductal cancer cell line, SW-1990. Incubation for up to 24 h with forskolin, an activator of adenylate cyclase, or isobutylmethyl xanthine, a theophylline analog, increased cellular cAMP levels by over 100-fold and significantly increased CEA release and cellular CEA content. Whereas cAMP levels were augmented within 10 min of exposure to these agents, CEA release and CEA cell content were not increased until 90 min and 24 h, respectively. Similar results were obtained using dibutyryl-cAMP, a cAMP analog, but not using sodium butyrate, a metabolite of dibutyryl-cAMP. Cells were incubated with 35S-cysteine and 3H-glucosamine in the presence or absence of forskolin in order to compare the effects of high cAMP levels upon the synthesis and release of total proteins, total glycoproteins, and immunoprecipitable CEA. Both CEA synthesis and release were enhanced by forskolin, but these effects were not specific to CEA since the release of labeled proteins and glycoproteins also increased. In addition, altered CEA expression caused by forskolin was consistently associated with a cessation of cell division, an effect which was reversible upon removing the agent. There was no effect upon cell morphology or viability. The data indicate that increased levels of cellular cAMP in pancreatic cancer cells is associated with decreased cell proliferation and increased expression of CEA and other glycoproteins.

Effects of cyclic adenosine and cyclic guanosine nucleotides and theophylline on gastrin synthesis and secretion in rat antral organ culture.

The effect of cyclic adenosine and cyclic guanosine nucleotides and theophylline upon gastrin synthesis and secretion were examined in rat antral mucosa maintained in organ culture. In concentration-response experiments, cyclic AMP, dibutyryl cyclic AMP and theophylline stimulated gastrin release and incorporation of [3H]-tryptophan into gastrin: a high degree of positive correlation was demonstrated between gastrin synthesis and gastrin release in response to control and test culture conditions. Cyclic GMP and dibutyryl cyclic GMP also stimulated gastrin secretion to a degree similar to that seen with cyclic AMP. These studies provide support for the proposal that the cyclic nucleotide system is involved in the stimulation of gastrin synthesis and release by the antral gastrin cell.

Effect of cyclic adenosine 3',5'-monophosphate antagonists on endotoxin-induced inhibition of human neutrophil chemotaxis

We reported previously that Escherichia coli endotoxin inhibited human neutrophil chemotaxis toward C5a. This effect of endotoxin was antagonized by anti-inflammatory steroids. We now report that dibutyryl cyclic adenosine 3',5'-monophosphate, prostaglandin E1, isoproterenol, and cholera toxin also antagonize the suppression of chemotaxis by endotoxin. Each compound inhibited the effect of endotoxin in a dose-dependent fashion. To be effective, each compound except cholera toxin had to be present at the time of endotoxin challenge. Furthermore, propranolol blocked the protective effect of isoproterenol against endotoxin but not the protective effect of dibutyrl cyclic adenosine 3',5'-monophosphate or prostaglandin E1. Dibutyryl cyclic guanosine 3',5'-monophosphate, adenosine 5'-monophosphate, phenylephrine, prostaglandin F2 alpha, and carbachol did not modify the suppression of chemotaxis by endotoxin. Anti-inflammatory steroids and dibutyryl cyclic adenosine 3',5'-monophosphate are thought to stabilize phospholipids in certain cell membranes. This phospholipid-stabilizing action may contribute, at least in part, to the protective effect against endotoxin-mediated suppression of neutrophil chemotaxis.

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.

Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3',5'-monophosphate

The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.

Micropuncture Studies of the Motility of Rete Testis and Epididymal Spermatozoa

The effect of cyclic adenosine 3':5'-monophosphate (cAMP) and caffeine on the motility of spermatozoa obtained in vivo by micropuncture from the rat rete testis, caput epidiymidis, and cauda epididymidis was studied. Spermatozoa from all sites were immobile in their native fluid. Rete testis spermatozoa were not motile under any experimental conditions. After dilution in salt solution, some caput sperm exhibited circular motion, whereas most cauda sperm swam progressively. Dextrose enhanced the motility of sperm from both epidiymal sites. Caffeine further increased the motility of epididymal sperm. Dibutyryl cAMP and cAMP stimulated caput spermatozoa but had no effect on cauda spermatozoa.

The effect of cyclic adenosine 3?,5?-monophosphate on the uptake of estradiol by the neonatal mouse uterus

Uterine tissue from neonatal mice was incubated in vitro at 0 degrees C for 1 h in a medium containing 1 X 10(-8) M 3H-estradiol with or without 1 X 10(-4) M dibutyryl cyclic AMP. In some incubations the temperature was raised to 37 degrees C for 15 min after incubation in the cold, in others the temperature was kept at 0 degrees C during this 15 min period. The tissue was frozen in liquid propane cooled in liquid nitrogen, sectioned at 2 or 4 microns, and autoradiograms prepared according to the dry-mount procedure. cAMP increases the cellular uptake of 3H-estradiol in uterine tissue. After rising the temperature to 37 degrees C, grains appeared over the nuclei. cAMP at low temperature increased the cellular uptake of 3H-estradiol, but the grains were not associated with the nuclei. In the autoradiograms the grain number above the epithelium was markedly less than above the stroma.

The effect of cyclic adenosine- and guanosine 3′,5′-monophosphate on the normal and drug-increased coronary blood flow

Cyclic 3′,5′-AMP (cAMP), cyclic 3′,5′-GMP (cGMP) and their more lipo-phylic derivatives (cDBA, cMBA, cDBG) were studied in conscious, resting dogs prepared with chronically implanted electromagnetic flow probes and catheters. Coronary blood flow (CBF), heart rate, aortic blood pressure and in some cases, cardiac output were recorded. cAMP, cDBA and cMBA increased the normal and pharma-cologically elevated CBF while cGMP and its dibutyryl derivative were without effect. Pretreatment with theophylline, a phosphodiesterase inhibitor, diminished the effect of cAMP but enhanced the effect of cDBA and cMBA. The data suggest that cAMP cannot be considered as a second messenger responsible for the maintenance of coronary vascular tone. It may, however, be responsible for coronary vasodilation.

The Effect of Cyclic Adenosine 3', 5' Monophosphate, Methyl Xanthines and Nicotinic Acid on Plasma Nonesterified Fatty Acids in Sheep

The intravenous injection of 3', 5' AMP, 10 mg/kg, caused an immediate rise in blood glucose in normal sheep. In sheep where liver glycogen was depleted by pretreatment with 100 mg/kg of nicotinic acid, the blood glucose response was greatly reduced. The glycogenolytic effect of 3', 5' AMP was thus easily demonstrated. The injection of 3', 5' AMP was followed by an immediate rise in NEFA, which was most pronounced in the pretreated animals. After the initial increase there was a fall in NEFA in both the normal and the pretreated animals. The role of hyperglycemia for the NEFA response to exogenous 3', 5' AMP was discussed. It seemed possible that hyperglycemia may modify the initial increase in NEFA. The later fall in NEFA was discussed as a complex response, which may be the result of the action of several hormones, which may be released by 3', 5' AMP injection. Nicotinic acid and pyridyl-3-acetic acid showed a potent antilipolytic activity. Nicotinic acid caused a rebound elevation of NEFA. No rebound was observed after pyridyl-3-acetic acid. Pyridyl-3-acetic acid, however, did not change NEFA when administered orally. Pyridyl-2-acetic acid had no antilipolytic effect. Of the methyl xanthines caffeine and theophylline showed about the same lipolytic activity. Theobromine was quite ineffective.

Effect of cyclic adenosine 3',5'-monophosphate on the incorporation of 3H-leucine into polypeptides by beef thyroid polysomes in vitro.

Polysomes were isolated from beef thyroids and then analyzed in a cell-free system in order to determine whether adenosine 3′,5′- cyclic monophosphate (cAMP) was capable of eliciting a direct effect on protein synthesis. When concentrations of ATP and GTP were present which resulted in optimal incorporation of 3H-leucine into protein in vitro (i.e., 1 and 1.6 mM, respectively), addition of cAMP to the cellfree system did not cause an increase in protein synthesis. On the other hand, when suboptimal amounts of GTP were used in the system, addition of cAMP resulted in a slight stimulation of 3H-leucine incorporation. Similarly, if ATP was omitted from the incubation mixtures, cAMP caused up to a 2-fold increase in the rate of protein synthesis. Under optimal conditions for protein synthesis, a decrease in incorporation was observed by the addition of cAMP, dibutyryl cAMP or 5′-AMP. This fact, together with the removal of inhibition by cAMP in the presence of theophylline, suggests that degradation products ...

Immunofluorescence studies on the effect of cyclic adenosine 3',5'-monophosphate in tissue cultue preparations of the cervicovaginal anlage from neonatal mice.

The authors have studied the production of antigenic material, specific for the cervicovaginal epithelium, in organ cultures of the cervicovaginal region from neonatal mice. In control cultures the antigenic material appeared at a time corresponding to the normal appearance under in vivo conditions. When cyclic adenosine 3′, 5′-monophosphate (dibutyryl derivate) was added to the medium, a strong increase of the fluorescent material was seen.

The Effect of Cyclic Adenosine 3', 5'-Monophosphate on Carbohydrate Metabolism of Rats

The Effect of Cyclic Adenosine 3', 5'-Monophosphate on Carbohydrate Metabolism of Rats