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PubMed1991

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12.

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.

Amino Acid Sequence↗
PubMed1986

The roles of cAMP and cAMP-dependent protein kinase in the regulation of adrenocortical functions: analysis using DNA-mediated gene transfer.

DNA-mediated gene transfer was used to evaluate the cause and effect relationship between mutations in cAMP-dependent protein kinase activity and cellular resistance of adrenocortical tumor cells to ACTH and cAMP. Protein kinase defective, Kin 8 adrenocortical tumor cells were transformed with genomic DNA from an ACTH- and cAMP-responsive adrenocortical cell line and screened for the recovery of morphological responses to the cAMP analog 8-bromo-cAMP (8BrcAMP). 8BrcAMP-responsive transformants were recovered with a frequency of approximately 0.5 per 10(3) transformation-competent cells. These transformants recovered the ability to round up in the presence of ACTH and were able to respond to both ACTH and 8BrcAMP with increased steroidogenesis. They also recovered cAMP-dependent protein kinase activity. The transformants, however, were unstable and concomitantly lost cAMP-dependent protein kinase activity and steroidogenic and morphological responses to ACTH and 8BrcAMP. These observations suggest that a single gene, probably the gene encoding the regulatory subunit of cAMP-dependent protein kinase, is responsible for the resistance of the Kin 8 mutant to ACTH and cAMP.

Adrenal Cortex Neoplasms↗
PubMed1992

Bovine tracheal serous cell secretion: role of cAMP and cAMP-dependent protein kinase.

The role of adenosine 3',5'-cyclic monophosphate (cAMP) and protein phosphorylation during beta-adrenergic receptor stimulation of bovine tracheal gland serous cells was investigated in vitro. Isoproterenol, a beta-adrenergic agonist, increased the secretion of 35S-labeled molecules. Intracellular cAMP levels were increased within 1 min after stimulation of bovine tracheal gland serous cells with isoproterenol. The dose-response relationship for isoproterenol-stimulated generation of cAMP correlated with the dose-response relationship for isoproterenol-stimulated secretion of 35S-labeled molecules. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated both isoproterenol-evoked secretion of 35S-labeled molecules and the production of intracellular cAMP, and the beta-adrenergic receptor antagonist propranolol completely blocked both effects. The secretory response of the cells to isoproterenol could be mimicked by the cAMP analogues 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate. Activity of cAMP-dependent kinase was measured in soluble and particulate cell extracts. cAMP effected the state of phosphorylation of proteins associated with the soluble but not the particulate fraction. These studies are consistent with the hypothesis that beta-adrenergic stimulation of secretion from bovine tracheal gland serous cells occurs via a cAMP-mediated pathway and that one of the molecular events in this pathway is cAMP-dependent protein phosphorylation.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Evidence for compartmentalization of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases in rat Leydig cells using site-selective cAMP analogs.

It is generally believed that LH-stimulated steroidogenesis in Leydig cells is via activation of the cAMP second messenger system. The present study used cAMP analogs selective in their binding to the two types of cAMP-dependent protein kinase (PK-A) and/or selective in their binding to the two cAMP-binding sites on the regulatory subunits of PK-A. As the two cAMP-binding sites show positive cooperativity, synergistic increases in androgen production by rat Leydig cells occurred when they were incubated in vitro with analog pairs that selectively activated PK-A type I or II. This confirmed that both types of PK-A are present in Leydig cells and demonstrated that both PK-A types are capable of activating steroidogenesis. Synergistic increases in androgen production were also observed when either a type I selective analog (8-aminohexylamino-cAMP) or a type II selective analog (8-thiomethyl-cAMP) was paired with added cAMP. However, when these analogs were paired with LH or the adenylate cyclase activator forskolin, a synergistic increase in steroidogenesis occurred only with the type I selective analog. These results suggest that PK-A type I is compartmentalized in Leydig cells so that it has preferential access to endogenously produced cAMP.

Animals↗
PubMed1984

Effect of synthetic 1-34 fragment of human parathyroid hormone on plasma adenosine 3',5'-monophosphate (cAMP) concentrations and the diagnostic criteria based on the plasma cAMP response in Ellsworth-Howard test.

The effect of synthetic 1-34 fragment of human parathyroid hormone (hPTH(1-34] on plasma adenosine 3',5'-monophosphate (cAMP) in human subjects and the diagnostic criteria for the plasma cAMP response in an Ellsworth-Howard test were studied. 20 or 30 micrograms hPTH(1-34) and 200 USP Parathormone (Eli Lilly & Co.), infused intravenously over 5 min, produced very similar patterns of response in plasma cAMP, peak values being observed within 5 or 10 min after the end of the infusion. The maximum levels of plasma cAMP were over 111.5 pmol/ml in all of the normal subjects (n = 5) and patients with idiopathic hypoparathyroidism (n = 22), including those of children, but the plasma cAMP did not rise above 65.0 pmol/ml in pseudohypoparathyroidism (n = 7). There existed a significant correlation between the maximum plasma cAMP concentrations and increases in urinary cAMP excretion after infusions of both hPTH(1-34) and Parathormone. These results suggest that hPTH(1-34) has effects essentially identical to those of native PTH on plasma cAMP. We would like to propose a new diagnostic criterion in the Ellsworth-Howard test: a peak value of plasma cAMP over 100 pmol/ml after 30 micrograms hPTH(1-34) infusion is regarded as a normal response.

Adolescent↗
PubMed1991

cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity.

The theme of this study is an evaluation of the involvement of cAMP and cAMP-dependent protein kinase (PKA) in the regulation of the human heat shock protein (hsp) 70 gene promoter. Expression of a highly specific protein inhibitor of PKA (pRSVPKI) inhibited the basal as well as heat- and cadmium-induced expression of the cotransfected pHBCAT, a human hsp 70 promoter-driven reporter gene; this inhibition was dependent on the amount of pRSVPKI used. The effect of an expression vector of the RI regulatory subunit of PKA, pMTREV, was similar to that of pRSVPKI; pMTREV inhibited both the basal as well as the heat-induced expression of pHBCAT. The specificity of effects of these expression vectors was demonstrated by the lack of effect of a mutant PKI gene and by the unaffected expression of a reference gene (pRSV beta gal) under these conditions. Analysis of the effects of dibutyryl cAMP (1 mM), forskolin (10 microM), and 8-Br-cAMP (1 mM) on the transient expression of pHBCAT showed that these cAMP-elevating agents stimulated the hsp 70 promoter activity, whereas cAMP (1 mM) was without effect. Chloramphenicol acetyltransferase gene constructs with truncated or mutated hsp 70 promoter were used to define the cis-acting DNA element(s) that confer this cAMP stimulation; the heat induced (42 degrees C) expression was used as a control. Mutation of the adenovirus transcription factor element (pLSN-40/-26) greatly reduced the basal level of expression; forskolin had little or no effect on this adenovirus transcription factor-minus promoter, although the promoter activity was very heat inducible. The absence of a functional heat shock consensus element (HSE) in the construct pLSPNWT rendered the promoter heat insensitive; this construct was forskolin responsive although the magnitude of this stimulation was reduced when compared with that of a control construct with HSE. These results were corroborated by studies using consensus sequence of ATF (ATFE) and HSE as competitors to titrate our cellular factors that may interact with these elements. We showed that cotransfection with ATFE and HSE depressed the basal (37 degrees C) expression of pHBCAT by 25 and 60%, respectively. The heat-induced expression of pHBCAT was not significantly affected by the cotransfection of ATFE and was reduced by 60% when HSE was cotransfected. ATFE and HSE reduced the forskolin-induced pHBCAT expression by 70 and 40%, respectively. The implications of these findings as they relate to the action of cAMP and cAMP-dependent protein kinase in the control of heat shock gene expression are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1976

Influence of adenosine, cAMP and db-cAMP on responses of the isolated terminal guinea-pig ileum to electrical stimulation.

Adenosine, cAMP and db-cAMP were found to inhibit nerve-mediated contractions of the terminal ileum elicited by electrical stimulation (ES). Responses to ES at 30 Hz, which are believed to be of adrenergic origin, were considerably more affected than the responses to ES at 3 Hz, which may be of purinergic orign. The following order of relative inhibitory potencies was established: adenosine greater than cAMP greater than db-cAMP. Adenosine, cAMP and db-cAMP did not affect the contractile responses of the preparation to histamine, potassium and noradrenaline. Therefore, the observed inhibition of nerve-mediated contractions was thought to be due to an action on presynaptic sites, i.e. on the neural network within the intestine. This action was antagonized by an increase in external calcium and by aminophylline, which is known to increase intracellular calcium. The present experiments suggests that the inhibitory action of adenosine, cAMP and db-cAMP results from a reduction of calcium fluxes across the neural membrane and to a subsequent reduction in the release of neurotransmitter(s). The inhibitory effect of cAMP appeared to be nonspecific and was presumably related to adenosine in its molecule.

Adenosine↗
PubMed1990

A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP.

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.

Animals↗
PubMed1985

Kinetics and concentration dependence of reversible cAMP-induced modification of the surface cAMP receptor in Dictyostelium.

We have previously reported that extracellular cAMP induced a reversible shift, from apparent Mr = 40,000 to 43,000, in the electrophoretic mobility of a polypeptide identified by photoaffinity labeling with [32P]8-N3-cAMP as the cAMP receptor of Dictyostelium (Klein, P., Theibert, A., Fontana, D., and Devreotes, P. (1985) J. Biol. Chem. 260, 1757-1764). In this report, we examine the kinetics and concentration dependence of this stimulus-induced receptor modification. Prior to stimulation, 90% of the receptors migrated as the higher mobility form (Mr = 40,000) and 10% as the lower mobility form (Mr = 43,000). Following 15 min of persistent stimulation with 1 microM cAMP, the per cent of receptors migrating as the lower mobility form rose to 80%. This transition occurred with a half-time of 2.5 min. Removal of the stimulus initiated a return to the basal state which occurred with a half-time of about 6 min at 22 degrees C. No reversal occurred at 0 degrees C. Addition and removal of a 50 nM cAMP stimulus induced transitions with similar kinetics, but the final plateau value reached was only 40% lower mobility form. The stimulus concentration which induced 50% of the maximal transition from higher to lower mobility forms at steady state was 27 nM, similar to the KD for [3H]cAMP binding. Scatchard analysis of [3H]cAMP binding indicated that, although a 20% down-regulation occurs during cAMP stimulation, there is no significant difference in the affinities of the higher and lower mobility forms of the receptor. The unoccupied higher and lower mobility forms of the receptor, designated R and D, are considered to be in rapid equilibrium with liganded forms, designated RL and DL. The rate constants for interconversion of the receptor forms R (Formula: see text) D and RL (Formula: see text) DL were calculated from the kinetic data: k1 = 0.012, k-1 = 0.104, k2 = 0.222, and k-2 = 0.055. The interconversion steps are not at equilibrium, suggesting that an energy expenditure occurs during the receptor modification. The pattern of modulation of the cAMP-induced receptor modification suggests that it may be the biochemical mechanism of adaptation.

Adaptation, Physiological↗
PubMed1986

Identification of a ternary complex between cAMP and a trimeric form of cAMP-dependent protein kinase.

One of the intermediates involved in dissociation and reassociation of the subunits of the type II cAMP-dependent protein kinase has been characterized. This intermediate can be generated when the protein kinase is prepared from the isolated catalytic subunit (C) and the isolated regulatory subunit-[3H]cAMP complex (R2-[3H]cAMP4) by dialysis for 18 h followed by gel filtration. The intermediate, which could be separated from the holoenzyme and the isolated subunits by polyacrylamide gel electrophoresis, had an apparent molecular weight of 149,000, consistent with an R2C form. Following electrophoresis, measurements of R and bound nucleotide indicated that R2C was half-saturated with [3H]cAMP. The bound [3H]cAMP exhibited biphasic dissociation kinetics indicating that both types of cAMP binding sites were occupied. These findings suggested that the intermediate is R2C-cAMP2. This intermediate was not seen when the dialysis time was increased to 5 days, but could be observed when cAMP was added to the holoenzyme or when holoenzyme was mixed with R2cAMP4 and cAMP. The presence of two occupied cAMP binding sites on this intermediate suggests that there is minimal cooperativity between the two members of the regulatory subunit dimer, i.e. one member of the dimer binds 2 molecules of cAMP while the other binds C.

Animals↗
PubMed1983

Regulation of cAMP-dependent protein kinase subunit levels in Friend erythroleukemic cells. Effects of differentiation and treatment with 8-Br-cAMP and methylisobutyl xanthine.

Friend erythroleukemic cells contain both type I and type II cAMP-dependent protein kinases. The amounts of type I and type II regulatory subunits (R I and R II) were established by a combination of the equilibrium binding of cyclic [3H]AMP and specific immunoprecipitation. In control cells the total specific cAMP-binding activity was 9.8 pmol/mg of cytosolic protein and the ratio of R II to R I was 1.2. After the induction of differentiation by growth in 2% dimethyl sulfoxide, the total cAMP-binding capacity doubled, but larger and opposing changes in R II and R I levels were observed. The concentration of R II tripled (to 16.6 pmol/mg) while R I content (1.5 pmol/mg) declined to one-third of the control cell level, resulting in an R II:R I value of 11.1. Treatment of Friend cells with 0.5 mM 8-bromoadenosine 3':5'-monophosphate (8-Br-cAMP) and 0.2 mM methylisobutyl xanthine for 2 days did not stimulate cell differentiation. however, the cAMP-binding capacity nearly doubled and the cells accumulated a high level of R II while R I declined. This resulted in a distribution of R subunits closely resembling that of differentiated cells. Cyclic AMP-stimulated histone kinase activity increased in proportion to cAMP-binding activity during differentiation; no such increase was seen following 8-Br-cAMP treatment. Fractionation of cytosolic extracts by DEAE-cellulose chromatography revealed that both R I and R II were fully associated with the catalytic subunit (C) of protein kinase before and after differentiation. In extracts of 8-Br-cAMP-treated cells all of R I and 40 to 60% of R II were not associated with C. Free R II isolated from these cells was indistinguishable from RII present in holoenzyme. Thus, erythroid differentiation of Friend cells results in a coordinate accumulation of R II and C subunits as well as a decline in R I content. In contrast, chronic exposure to 8-Br-cAMP elicits similar changes in R subunit levels but disrupts the coordinate regulation of R and C content.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Binding of cAMP derivatives to Dictyostelium discoideum cells. Activation mechanism of the cell surface cAMP receptor.

The binding of 16 derivatives of cAMP to the surface of Dictyostelium discoideum cells was analyzed. The binding affinity is strongly reduced (more than 14.5 kJ/mol) if a cAMP derivative is no longer able to form a hydrogen bond at N6H2, or at O3'. Decreasing polarity of the base moiety is closely correlated to increasing binding affinity to the cAMP receptor (r = 0.98, p less than 0.1%). Based on these results we propose that cAMP is bound to the receptor via hydrogen bonds at N6H2 and O3', and that the adenine moiety is bound in a hydrophobic cleft of the receptor. A stereospecific interaction between the receptor and the phosphate moiety of cAMP has not been observed. The first detectable response of D. discoideum cells to cAMP is an increase of intracellular cGMP levels. A close correlation exists between the binding affinity of the cAMP derivatives and the potency to induce a cGMP response (r = 0.97, p less than 0.01%). There are two exceptions; both derivatives are modified in the exocyclic oxygen atoms of the phosphate moiety (sulfur in equatorial position or dimethylamino in apical position). These derivatives bind with approximately the same affinity as their stereoisomers (respectively sulfur in apical or dimethylamino in equatorial position), but in contrast to their stereoisomers, they do not induce elevations of cGMP levels. This suggests that, in addition to the binding interactions mentioned above, activation of the receptor requires a stereospecific interaction between the receptor and the phosphate moiety of cAMP. Quantum-chemical calculations by Van Ool and Buck (Van Ool, P. J. J. M., and Buck, H. M. (1982) Eur. J. Biochem. 121, 329-334) suggest that this activating interaction is a covalent bond between cAMP and the receptor.

Binding, Competitive↗
PubMed2006

Mental health needs of children and adolescents at camp: are they being assessed and treated appropriately by the camp nurse?

TOPIC: Increasingly, more children and adolescents are attending camps with mental health concerns. This can pose a challenge for camp nurses who may lack experience in assessment and treatment of mental health issues. PURPOSE: To focus on the importance of addressing and treating mental health needs of children and adolescents at camp utilizing the Scope and Standards of Psychiatric Mental Health Nursing Practice. SOURCES: Personal observations, camp nursing experience, and scholarly published literature. CONCLUSIONS: It is paramount that mental health needs of children and adolescents at camp are addressed and managed appropriately by the camp nurse. Education of camp nurses and camp administrators is also a vital part of providing care.

Adaptation, Psychological↗
PubMed1995

Characterization of the isolated cAMP-binding B domain of cAMP-dependent protein kinase.

A 14.4-kDa cAMP-binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP-dependent protein kinase type I alpha regulatory subunit (RI alpha). The full-length RI alpha from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser-252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RI alpha, the [3H]cAMP exchange rate was comparable to that of the B domain of full-length RI alpha containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high-affinity cAMP-binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2-mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP-binding domain can function independently of any other domain structures of the R subunit.

Amino Acid Sequence↗
PubMed2000

Modeling the cAMP-induced allosteric transition using the crystal structure of CAP-cAMP at 2.1 A resolution.

After an allosteric transition produced by the binding of cyclic AMP (cAMP), the Escherichia coli catabolite gene activator protein (CAP) binds DNA specifically and activates transcription. The three-dimensional crystal structure of the CAP-cAMP complex has been refined at 2.1 A resolution, thus enabling a better evaluation of the structural basis for CAP phenotypes, the interactions of cAMP with CAP and the roles played by water structure. A review of mutational analysis of CAP together with the additional structural information presented here suggests a possible mechanism for the cAMP-induced allostery required for DNA binding and transcriptional activation. We hypothesize that cAMP binding may reorient the coiled-coil C-helices, which provide most of the dimer interface, thereby altering the relative positions of the DNA-binding domains of the CAP dimer. Additionally, cAMP binding may cause a further rearrangement of the DNA-binding and cAMP-binding domains of CAP via a flap consisting of beta-strands 4 and 5 which lies over the cAMP.

Allosteric Regulation↗
PubMed1989

Conformational changes of type II regulatory subunit of cAMP-dependent protein kinase on cAMP binding.

The effect of cAMP on the conformation of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from bovine heart was investigated by UV-difference and circular dichroism (CD) spectroscopy. The UV-difference spectrum of RII with and without cAMP showed a positive band around 278 nm and a negative band at 256 nm. Similarly, cAMP enhanced the ellipticity of RII in the region between 280 and 300 nm and decreased that between 250 and 280 nm. In addition, cAMP transformed the far-UV CD spectrum of RII from that of a negative band minimally at 209 nm with a shoulder at 223 nm to one with two minima at 222 and 211 nm. These data show that cAMP induces conformational changes of RII upon binding. Such structural changes may be the basis of activation of cAMP-dependent protein kinases by cAMP.

Animals↗
PubMed1993

Prostaglandin E2, cAMP and cAMP-dependent protein kinase isozymes during decidualization of rat endometrial stromal cells in vitro.

When endometrial stromal cells from rat uteri sensitized for the decidual cell reaction are cultured in vitro, they undergo decidualization, as indicated by increased alkaline phosphatase (ALP) activity. Prostaglandin E2 (PGE2) stimulates this increase in activity. To determine the role of cAMP in the stimulation, we examined the effect of 2':5'-dideoxyadenosine (DDA), an inhibitor of adenylate cyclase, on the ability of PGE2 to increase ALP activity. As indicated by [3H]cAMP accumulation in endometrial stromal cells preincubated with [3H]adenine, DDA inhibited PGE2-stimulated synthesis of cAMP in a concentration-dependent manner. Furthermore, DDA caused a significant decrease in the PGE2-induced ALP activity on day 3 of culture. Dibutyryl cAMP overrode this inhibition. The effect of DDA was not mimicked by adenosine, which had a stimulatory effect on ALP activity in the non-stimulated cultures and no significant effect in PGE2-stimulated cultures. Thus the inhibitory effect of DDA on PGE2-stimulated ALP activity is unlikely to be mediated by adenosine-related receptors. These results suggest that cAMP is an essential, but not necessarily the only, intracellular messenger of PGE2 in endometrial stromal cells during decidualization. The isozymes of cAMP-dependent protein kinase (PKA) mediating the effect of cAMP were assessed by using cAMP analogues directed at selective sites of PKA isozymes. Synergistic activation of ALP activity in endometrial stromal cells by pairs of analogues directed at types I and II PKA suggested that both types were functionally important during decidualization.

Adenosine↗
PubMed1991

Prostaglandin E2 induction of monoblastic differentiation utilizes both cAMP and non-cAMP dependent signalling systems.

U937 cells can be induced to express receptor for complement 5a (C5aR) by sequential 2 day treatments of cells with dihydroxyvitamin D-3 (1,25(OH)2D3) followed by prostaglandin E2. We asked whether the action of prostaglandin E2 to cause maximal C5aR expression required only activation of the cAMP-dependent protein kinase (PKA). Prostaglandin E2 dose dependently activated PKA in control and 1,25(OH)2D3 treated cells; by 4 h the PKA did not respond to further prostaglandin E2 challenge. We hypothesized that prostaglandin E2 actions transduced via PKA should be complete by 4 h; i.e., C5aR induction should be equivalent in cells treated with prostaglandin E2 for 4 h and for 2 days. All cells were treated for the first 2 days with 1,25(OH)2D3 and the second 2 days with prostaglandin E2 or cAMP analogs. C5aR number was measured after 4 days total culture. 4 h pulse treatments with agents were given at the end of the 1,25(OH)2D3 treatment. Cells exposed to a 4 h pulse of prostaglandin E2 had only 68.2 +/- 4.4% the amount of C5aR seen in cells continuously exposed to prostaglandin E2. Continuous culture with a cAMP analog pair (50 microM each of 8-thiomethyl-cAMP + N6-benzoyl-cAMP), which caused a 41.7% +/- 10.8% increase PKA activation above basal, resulted in only 51% +/- 16% of the C5aR numbers seen in cells cultured for 2 days with prostaglandin E2, where PKA remained at basal activity. We therefore concluded that C5aR expression caused by prostaglandin E2 could not be ascribed entirely to duration or degree of activation of cAMP-dependent signalling pathways. We investigated the possibility that the calcium sensitive protein kinase C was involved. Cytoplasmic protein kinase C was increased 154% +/- 14% above control in cells treated with sequential 2 days treatments of 1,25(OH)2D3 and prostaglandin E2. A 147% +/- 2% increase in membrane associated protein kinase C was also seen 10 min after phorbol myristate acetate stimulation in the above treatment group. Finally, phorbol myristate acetate augmented the C5aR induction caused by cAMP analog. We propose that the mechanism of prostaglandin E2 synergism with 1,25(OH)2D3 in causing C5aR induction in U937 cells includes signal transduction not only by the cAMP cascade, but also via protein kinase C modulated pathways.

Calcitriol↗