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PubMed1987

Forskolin's effect on transient K current in nudibranch neurons is not reproduced by cAMP.

Forskolin, a diterpene extracted from Coleus forskolii, stimulates the production of cAMP in a variety of cells and is potentially an important tool for studying the role of cAMP in the modulation of neuronal excitability. We studied the effects of forskolin on neurons of nudibranch molluscs and found that it caused characteristic, reversible changes in the amplitude and waveform of the transient K current, IA, and also activated an inward current similar to the cAMP-dependent inward current previously described in molluscan neurons. Forskolin altered the time course of IA activation and inactivation but did not affect the voltage dependence or the reversal potential of the current. IA normally inactivates exponentially, but in forskolin the time course of inactivation can be fit by the sum of 2 exponentials with an initial rate that is faster than the control and a final rate that is much slower. On depolarization in forskolin, IA begins to activate at the normal rate, but a slower component of activation is also seen. The changes in IA in the nudibranch cells were qualitatively different than the changes caused by forskolin in Aplysia bag cell neurons (Strong, 1984). Experiments were performed to determine whether these effects of forskolin require cAMP. Intracellular injection of cAMP, application of membrane-permeable analogs of cAMP, application of phosphodiesterase inhibitors, and intracellular injection of the active catalytic subunit of cAMP-dependent protein kinase did not affect the amplitude or waveform of IA. Also, the changes in IA that are caused by forskolin were not prevented or reversed by intracellular injection of an inhibitor of cAMP-dependent protein kinase. Cyclic AMP did, however, activate inward current at voltages near the resting potential. We conclude that the changes in IA and the activation of inward current represent separate affects of forskolin. The inward current appears to depend on an increase in intracellular cAMP, while the changes in IA do not. These experiments show that, in addition to activating adenylate cyclase, forskolin may have a separate direct affect on the transient K current.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450(17 alpha) by an androgen receptor-mediated mechanism.

The regulation of the novo synthesis of the microsomal cytochrome P-450 enzyme, P-450(17 alpha), was studied in mouse Leydig cell cultures. Chronic treatment with 0.05 mM 8-Br-cAMP (cAMP) caused a time-dependent increase in 17 alpha-hydroxylase activity and in the amount of P-450(17 alpha), quantitated by immunoblotting. This increase in both activity and amount was enhanced by inhibiting testosterone production with aminoglutethimide, an inhibitor of cholesterol side-chain cleavage or SU 10603, an inhibitor of 17 alpha-hydroxylase. To examine the mechanism by which cAMP or cAMP plus inhibitors of testosterone production increased the activity and amount of P-450(17 alpha), changes in the rate of de novo synthesis were studied by measuring [35S]methionine incorporation into newly synthesized protein. Treatment with cAMP plus aminoglutethimide or SU 10603 caused a 2-fold or greater increase in the rate of de novo synthesis of P-450(17 alpha) compared to treatment with cAMP only. The addition of exogenous testosterone reversed this increase in the rate of synthesis, indicating that testosterone modulates the extent of cAMP-stimulated induction of P-450(17 alpha). This negative effect of testosterone could be mimicked by the addition of the androgen agonist, mibolerone, and prevented by the addition of the antiandrogen, hydroxyflutamide. Neither estradiol nor dexamethasone had any effect on the synthesis of P-450(17 alpha). Studies on the degradation of newly synthesized P-450(17 alpha) demonstrated that testosterone had no effect on the decay of P-450(17 alpha) during the first 24 h but caused a significant increase in the rate of decay between 24 and 48 h. These data indicate that testosterone produced during cAMP induction of P-450(17 alpha) negatively regulates the amount of this cytochrome P-450 enzyme by two distinct mechanisms: by repressing cAMP-induced synthesis of P-450(17 alpha) by an androgen receptor-mediated mechanism and by increasing the rate of degradation of P-450(17 alpha). A model is proposed for the regulation of P-450(17 alpha) in Leydig cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

[System of cAMP-dependent phosphorylation in NIH 3T3 cells passing into the resting state].

The changes in the cAMP level and the activity of cAMP-dependent protein kinase in NIH 3T3 cells going into the resting state induced by cell cultivation in a medium with 0.5% serum were investigated. It was shown that under conditions when cells are going into the resting state, the slight increase in the intracellular concentration of cAMP observed within the first 24 hours after cell transfer onto a serum-deficient medium is correlated with a simultaneous decrease of the cAMP phosphodiesterase activity. A procedure was developed allowing to measure the activity of cAMP-dependent protein kinase in cell lysates in the presence of cAMP-independent protein kinases. The activity of cAMP-dependent protein kinase in the cells going into the resting state markedly increases (up to 5-fold) after 9 days as compared to that of dividing cells. This rise in activity is due to the decrease of the content of a thermostable protein inhibitor of cAMP-dependent protein kinase rather than to the elevated level of cAMP.

Animals↗
PubMed1984

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin.

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.

Adenylyl Cyclases↗
PubMed1984

Demonstration of receptor heterogeneity and affinity modulation by nonequilibrium binding experiments. The cell surface cAMP receptor of Dictyostelium discoideum.

The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.

Cell Membrane↗
PubMed1984

Site of insulin inhibition of cAMP-stimulated glycogenolysis.

Insulin antagonized the glycogenolytic actions of cAMP in isolated hepatocytes even in the presence of the phosphodiesterase inhibitors, 1-methyl-3-isobutylxanthine or d-4-(3-butoxy-4-methoxybenzyl)-2- imidizolidione . Stimulation of glucose release by 8-bromo-cAMP, which is not appreciably hydrolyzed by phosphodiesterase, was also inhibited by insulin in the presence of d-4-(3-butoxy-4-methoxybenzyl)-2- imidizolidione . This demonstrates that insulin can antagonize cAMP stimulation of glycogenolysis independent of possible effects of insulin on adenylate cyclase or phosphodiesterase. Under conditions where changes in cyclic adenine nucleotide concentrations (cAMP and 8-bromo-cAMP) were prevented, insulin depressed the 8-bromo-cAMP-stimulated protein kinase activity ratio. We conclude that phosphodiesterase activation is not required for insulin antagonism of cAMP-stimulated glycogenolysis. The effect of insulin under these conditions can be explained by an action of insulin on cAMP-dependent protein kinase independent of changes in cAMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1980

The role of agonists that activate adenylate cyclase in the control of cAMP metabolism and enzyme release by human polymorphonuclear leukocytes.

An inverse relationship between cAMP content and effector function is ascribed generally to immune and inflammatory cells. Previous reports imply, however, that human polymorphonuclear leukocytes (PMN) are less responsive than other inflammatory cells to adenylate cyclase (AC) agonists. We therefore examined the effects of isoproterenol, prostaglandin E1 (PGE1), adenosine, and histamine on the adenosine 3',5'-monophosphate (cAMP) content of PMN and on particle-stimulated lysosomal enzyme release. For comparison, the effect of AC agonists on the cAMP content of human peripheral lymphocytes was evaluated in parallel. Although potent stimuli for cAMP accumulation in lymphocytes, the AC agonists produced only marginal increases in the cAMP content of PMN; this difference in responsiveness was independent of agonist concentration or length of incubation. Inhibition of lysosomal enzyme release by the AC agonists was likewise marginal (< 20%). The addition of theophylline with isoproterenol produced additive inhibition without significant cAMP increases. Hydrocortisone, which caused a small increase in the cAMP content, markedly potentiated the effects of AC agonists on the cAMP level in PMN; the synergistic increases in cAMP were accompanied by additive effects on lysosomal enzyme release. It is concluded that human lymphocytes and PMN exhibit differential sensitivity to AC agonists and that this difference may provide a basis for the selective modulation of individual PMN- or lymphocyte-mediated events.

Adenylyl Cyclases↗
PubMed1981

Formation and turnover of triglyceride-rich vesicles in the chick liver cell. Effects of cAMP and carnitine on triglyceride mobilization and conversion to ketones.

Refractile cytoplasmic vesicles are formed in less than 10 h when chick liver cell monolayers are incubated with serum-free medium containing 0.9 mM oleate. These vesicles are identical in microscopic appearance to those formed in monolayers by de novo fatty acid synthesis (Tarlow, D. M., Watkins, P. A., Reed, R. E., Miller, R. S., Zwergel, E. E., and Lane, M. D. (1977) J. Cell Biol. 73, 332-353), but require about one-seventh the incubation time to achieve comparable size. After release from the cells by lysis in hypotonic medium, the vesicles can be isolated by flotation at 27,000 X g. Electron microscopy reveals that the isolated vesicles are rimmed by a membrane. Analysis of vesicles isolated from cells labeled with [14C]oleate or [14C]acetate showed that greater than 95% of their 14C content was in the form of triglyceride and that most cellular [14C]triglyceride was contained in the triglyceride-rich vesicles. Exposure of cells to dibytyryl-cAMP after removal of oleate from the medium caused the disappearance of triglyceride-rich vesicles within 36 h. In the absence of cyclic nucleotide, the vesicles persist. Consistent with this morphological change, dibutyryl-cAMP caused a 5.5-fold activation of the apparent rate of mobilization of cellular [14C]triglyceride from cells previously labeled with [14C]oleate. L-(--)-Carnitine alone had no effect; however, when added with dibutyryl-cAMP, cellular triglyceride mobilization was activated 7.4-fold. Although [14C]triglyceride was the principal 14C-labeled product secreted in the absence of cyclic nucleotide and comprised 90% of the total, [14C]acetoacetate and [14C] beta-hydroxybutyrate became major products when cells were treated with dibutyryl-cAMP. Thus, dibytyryl-cAMP activated ketogenesis from cellular [14C]triglyceride by 200-fold and when added with L-(--)-carnitine, by 400-fold. Cells containing triglyceride-rich vesicles labeled with [2-glyceryl-3H]triglyceride were generated by incubation with medium containing [2-3H]glycerol. A comparison of the rates of loss of cellular [1-oleoyl-14C- and [2-glyceryl-3H]triglyceride revealed that substantial re-esterification, i.e. recycling, of 14C-fatty acid released by lipolysis occurred. Under conditions where recycling of 3H label ws minimal, it was determined that 15% of the cellular [2-glyceryl-3H]triglyceride was secreted "en bloc," i.e. without prior lipolysis. En bloc secretion was not affected by dibutyryl-cAMP. The rate of lipolysis of vesicle-associated [2-glyceryl-3H]triglyceride was increased 2.2-fold in the presence of dibutyryl-cAmP. Chloroquine markedly inhibited the dibutyryl-cAMP-dependent lipolysis suggesting the participation of lysosomes in the mobilization of triglyceride-rich vesicles. Mechanisms are presented which could account for the effects of cAMP and carnitine on the turnover of vesicle triglyceride both at the level of lipolysis and the utilization of the released fatty acids by mitochondria...

Animals↗
PubMed1981

[The role of cAMP in the energy metabolism of human erythrocytes].

In human red blood cell there is complete adenyl cyclase--cAMP system. However, the role of this system and its connection with energy metabolism in red cell is not known. Hence, we studied the effects of cAMP and DB-cAMP on energy metabolism in human red cell. Blood was taken out from haematologically healthy volunteers. Red cells were isolated by differential centrifugation and washed three times in Tris-Ringer buffer (pH 7.4). The red cells suspensions (in the above buffer) were simultaneously incubated without additions (control) and with cAMP or DB-cAMP (5 mmol/L). Incubation temperature was 37 degrees C. The samples of suspensions for the extraction of metabolites and cofactors were taken at 0 and 180 minutes of incubation. The lactate, glucose, G6P, F6P, TP, ATP, ADP and AMP were determined in neutralized perchloric acid extracts by specific enzymatic methods. Results of our experiments show that cAMP and DB-cAMP significantly increase red cell glycolysis. Under the same conditions these nucleotides induce positive "cros-over" point at the phosphofructokinase (PFK) step of the glycolysis. The levels of adenine nucleotides (ATP, ADP, AMP) were unchanged through a whole period of incubation. We concluded that cAMP and DB-cAMP stimulates glycolytic process in red cells probably by allosteric activation of PFK.

Adenine Nucleotides↗
PubMed1983

Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores.

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.

Affinity Labels↗
PubMed1984

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors.

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.

Adenosine Triphosphate↗
PubMed1994

Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells.

We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.

Animals↗
PubMed1993

Evidence for the involvement of at least two distinct transcription factors, one of which is liver-enriched, for the activation of the phosphoenolpyruvate carboxykinase gene promoter by cAMP.

A detailed analysis of the promoter sequence requirements for the cAMP induction of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was undertaken. It was determined that the cAMP-responsive unit of this promoter consisted of two independently weak or inactive components; the typical cAMP response element (CRE) sequence located at position -85 and a region of the promoter extending from -300 to -230 which contained multiple binding sites for liver-enriched nuclear proteins. A previous study had indicated that multiple binding sites are critical for the activity of this latter region (Liu, J., Park, E. A., Gurney, A. L., Roesler, W. J., and Hanson, R. W. (1991) J. Biol. Chem. 266, 19095-19102). In the present study, we extend this observation by demonstrating that the activity of this region can be effectively substituted for by three copies of just one subsite present in that region. This would suggest that the binding of three molecules of a single liver-enriched factor is what forms this component of the cAMP response unit. The other component of the cAMP response unit, the CRE, has been shown previously to be bound by these same liver-enriched factors as well as by cAMP response element binding protein, leading to some debate as to the identity of the protein mediating the cAMP response through this element. By two different experimental approaches, we show that neither CCAAT/enhancer binding protein nor D-site binding protein is the likely mediator of the cAMP response through the CRE, while cAMP response element binding protein is a possible candidate.

Base Sequence↗
PubMed1993

Decreased sensitivity to cAMP in the in vitro generation of memory splenic cytotoxic T-lymphocytes from aged mice: role of phosphodiesterase.

The effects of increased intracellular concentration of cyclic AMP (cAMP) on cellular immune function of young and aged mice were examined by using an in vitro anti-influenza cytotoxic T-lymphocyte (CTL) assay. We found that increasing intracellular concentrations of cAMP with either the cAMP analog, N,6O2'-dibutyryl cAMP, or the beta adrenergic agonist, metaproterenol, significantly inhibited the in vitro development of memory CTL activity of young mice. The development of CTL activity of aged mice, however, was less sensitive to the effects of these agents. We found that the S-isomer of cAMPS, a cAMP analog resistant to the effects of PDE, caused equivalent inhibition of CTL activity in the young and aged mice. This suggested that the cause of the decreased cAMP sensitivity in aging was increased PDE activity. In subsequent experiments, we found that the PDE from T-cells of aged mice have higher KM and higher Vmax for cAMP than the PDE from T-cells of young mice. Because beta-agonists probably act as a counter-regulatory hormone at an inflammatory site, the decreased sensitivity to cAMP in aging may be a compensatory response to the age-related decline in immune function.

Aging↗
PubMed1993

Cell-permeable non-hydrolyzable cAMP derivatives as tools for analysis of signaling pathways controlling gene regulation in Dictyostelium.

A novel class of cAMP derivatives were tested for binding to surface cAMP receptors (CAR), protein kinase A (PKA), and cAMP-phosphodiesterase (PDE) and for induction of three classes of cAMP regulated genes in Dictyostelium discoideum. These derivatives carry sulfur substitutions for either the axial (Sp) or equatorial (Rp) exocyclic oxygen atoms, while further modifications were introduced to provide specificity for binding to either CAR or PKA, and/or to increase lipophilicity and render the derivatives membrane-permeable. All derivatives bind weakly to PDE and are almost not degraded during incubation with Dictyostelium cells. One cAMP derivative, 6-thioethyl-purineriboside 3',5'-monophosphorothioate, Sp-isomer (Sp-6SEtcPuMPS), fulfills the criteria for selective activation of PKA in vivo. The compound enters Dictyostelium cells and reaches an intracellular concentration of 1 microM, sufficient to activate PKA, at an extracellular concentration of 30 microM, which is insufficient to activate CAR. Expression of cAMP-regulated prespore and prestalk genes and the aggregative PDE gene are effectively induced by CAR agonists and very poorly by PKA agonists. Even Sp-6SEtcPuMPS is ineffective to induce gene expression. These data not only indicate that surface cAMP receptors are the first targets for cAMP-induced gene expression, but argue against direct induction of expression of these genes by cAMP-induced PKA activation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

The second messenger cAMP elicits eating by an anatomically specific action in the perifornical hypothalamus.

We have previously shown that a membrane-permeant analog of cAMP, 8-bromo-cAMP (8-br-cAMP), elicits a vigorous eating response when microinjected into the perifornical hypothalamus (PFH) or lateral hypothalamus (LH) of satiated rats, suggesting that increases in cAMP in these areas may be important in the neural control of eating. To determine the locus of this effect, we compared the ability of 8-br-cAMP (1-100 nmol/0.3 microl) to elicit eating after microinjection into the PFH, LH, or the following bracketing areas: the anterior and posterior LH, paraventricular nucleus of the hypothalamus, thalamus, and amygdala. 8-br-cAMP at 50 nmol elicited eating (>/=3.4 gm in 2 hr) exclusively in the PFH and LH. At 100 nmol, 8-br-cAMP elicited a larger response in these areas and elicited a smaller, more variable response in the thalamus. We similarly mapped the feeding-stimulatory effects of compounds that increase endogenous cellular cAMP in naive rats. Combined microinjection of matched doses (300 nmol) of 3-isobutyl-1-methylxanthine and 7-deacetyl-7-O-(N-methylpiperazino)-gamma-butyryl-forskolin was effective exclusively in the PFH, eliciting an average 2 hr food intake of 8.4 +/- 2.0 gm. Collectively, these results suggest that increases in cellular cAMP within a specific brain site, the PFH, may play a role in the neural stimulation of eating.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Regulation of macrophage phagocytosis of apoptotic cells by cAMP.

Regulation of macrophage capacity to remove apoptotic cells may control the balance of apoptotic and necrotic leukocytes at inflamed foci and the extent of leukocyte-mediated tissue damage. Although the molecules involved in the phagocytic process are beginning to be defined, little is known about the underlying regulatory and signaling mechanisms controlling this process. In this paper, we have investigated the effects of treatment of human monocyte-derived macrophages with PGs and other agents that elevate intracellular cAMP on phagocytosis. PGE2 and PGD2 specifically reduced the proportion of macrophages that phagocytosed apoptotic cells. Similar results were obtained with the membrane-permeable cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP but not with the cGMP analogue dibutyryl-GMP. Consistent with the observation that phagocytosis was inhibited by cAMP elevation, treatment of monocyte-derived macrophages with PGE2 resulted in rapid, transient increase in levels of intracellular cAMP. These effects were not due to nonspecific inhibition of monocyte-derived macrophage phagocytosis given that ingestion of Ig-opsonized erythrocytes was unaffected. Elevation of cAMP induced morphologic alterations indicative of changes in the adhesive status of the macrophage, including cell rounding and disassembly of structures that represent points of contact with substrate containing actin and talin. These results strongly suggest that rapid activation of cAMP signaling pathways by inflammatory mediators regulates processes that limit tissue injury and that modulation of cAMP levels represents an additional therapeutic target in the control of resolution of inflammation.

Apoptosis↗
PubMed1995

Phase I clinical study with 8-chloro-cAMP and evaluation of immunological effects in cancer patients

The site-selective cyclic AMP analogue 8-chloro-cAMP (8-Cl-cAMP) is able to inhibit the growth of a wide variety of cancer cell lines in vitro and in vivo. 8-Cl-cAMP has been extensively investigated as a new potential anticancer agent and, more recently, preclinical Phase I studies have been conducted in animal models to study its toxicity. We have conducted the first Phase I trial with 8-Cl-cAMP to define the maximum tolerated dose, toxicity, plasma drug levels, and immunological effects in patients with cancers refractory to standard treatments. We have administered 36 courses of 8-Cl-cAMP to 17 patients by continous i.v. infusion of the drug for 5 days/week for 2 weeks followed by a 1-week rest period. Six increasing dose levels, from 0.01 to 0.25 mg/kg/h, were explored. Drug plasma levels were determined and the expression of interleukin 2 receptor alpha amount of natural killer cells, and cytolytic activity against K562 cells were measured in peripheral blood lymphocytes. A grade 4 and a grade 3 increase in serum creatinine and a grade 2 increase in blood urea nitrogen observed in two patients were the dose-limiting toxicity. The maximum tolerated dose (0.2 mg/kg/h) determined a grade 1 increase in serum creatinine. An increase in calcium levels was observed in several patients. The 8-Cl-cAMP plasma concentrations obtained at the steady state were in the range previously shown to be effective for cancer cell growth inhibition in vitro. Interleukin 2 receptor alpha expression, natural killer cell number, and cytolytic activity from peripheral blood lymphocytes were markedly increased after 8-Cl-cAMP administration at all dose levels. In conclusion, at doses below the maximum tolerated dose, 8-Cl-cAMP was not toxic but reached plasma concentrations in the potential therapeutic range for growth inhibition. Moreover, 8-Cl-cAMP determined a marked biomodulatory effect and showed antitumor activity.

Journal Article↗