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PubMed1993

cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch.

The cAMP-dependent Na+ current (INa,cAMP) modulates excitability in many molluscan neurons. Rapid activation of INa,cAMP by cyclic nucleotide, its ion dependence, and its blockade by divalent cations resemble cyclic nucleotide-activated cation currents in vertebrate photoreceptors and olfactory receptors, where activation has been found to be independent of kinase activity. We tested the phosphorylation dependence of INa,cAMP in neurons of the feeding and locomotory networks of the predatory marine snail Pleurobranchaea. Identified neurons of pedal and buccal ganglia were axotomized for recording the INa,cAMP response to iontophoretic injection of cAMP under two-electrode voltage clamp. Intracellular injections of specific peptide inhibitor of protein kinase A had no blocking effects on activation of INa,cAMP by iontophoretic injection of cAMP. Inward single-channel currents were activated in excised inside-out patches during exposure to cAMP in salines without added ATP. Sodium was the major current carrying ion. Two distinct types of INa,cAMP channel activity were observed, where opening probability and open times differed, but conductance was similar, 36.7 pS. These observations suggest that INa,cAMP activation occurs by direct binding of cAMP to a regulatory site at the channel, rather than by phosphorylation.

Animals↗
PubMed1993

8-Cl-cAMP induces truncation and down-regulation of the RI alpha subunit and up-regulation of the RII beta subunit of cAMP-dependent protein kinase leading to type II holoenzyme-dependent growth inhibition and differentiation of HL-60 leukemia cells.

8-Cl-cAMP, a site-selective cAMP analog, induces growth inhibition in a variety of cell types of human cancer cell lines. This inhibitory effect of 8-Cl-cAMP was related to its ability to differentially regulate type I versus type II cAMP-dependent protein kinase. In the present study we demonstrated a unique mechanism of action of 8-Cl-cAMP in the regulation of these kinase isozymes in HL-60 human promyelocytic leukemia cells. High-performance liquid chromatography (HPLC) resolved various isoforms of protein kinase present in HL-60 cells. In control cells, type I protein kinase (PKI) comprised more than 90% and type II protein kinase (PKII) less than 10% of the total cAMP-stimulated kinase activity. Treatment with 8-Cl-cAMP (5 microM, 72 h) decreased PKI to a level below 30% of that in untreated control cells and markedly increased PKII composed of three peaks. Photoaffinity labeling/SDS-polyacrylamide gel electrophoresis of column fractions identified the molecular species of regulatory (R) subunits present in protein kinases. Control cells contained high levels of the 48-kDa protein (RI) that composed PKI and low levels of the 50-kDa RII associated with PKII. 8-Cl-cAMP treatment brought about a decrease in the 48-kDa RI along with an increased formation of the truncated 34-kDa RI associated with PKI and an increase in the 50-54-kDa species of RII associated with PKII. A similar protein kinase profile as that shown by 8-Cl-cAMP treatment was observed in cells infected with the human RII beta retroviral vector: the 48-kDa RI of PKI decreased and the 52- and 54-kDa RII associated with PKII increased as compared with uninfected control cells. However, unlike 8-Cl-cAMP treatment, RII beta retroviral vector infection brought about no increase in the 34-kDa-truncated RI but exhibited an increase in the free 48-kDa RI subunit. As the 48-kDa RI and the 50-kDa RII were present in control cells, the enhanced expression of the 52- and 54-kDa RII proteins was due to overexpression of the RII beta gene. We identified the 48-kDa RI as RI alpha, the 50-kDa RII as RII alpha, the 52-kDa RII as RII beta, and the 54-kDa RII as the phosphorylated form of either the RII alpha or RII beta subunit. In vivo labeling experiments using [3H]8-Cl-cAMP demonstrated that 8-Cl-cAMP enters cells and binds to both PKI and PKII.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Acquisition of Pseudomonas cepacia at summer camps for patients with cystic fibrosis. Summer Camp Study Group.

To assess the risk of acquisition of Pseudomonas cepacia by person-to-person transmission at cystic fibrosis summer camps, we conducted in 1990 a study at three camps attended by patients with cystic fibrosis who had P. cepacia infection and patients without P. cepacia infection but who were considered susceptible to infection. We obtained sputum or throat cultures from campers on their arrival at, weekly during, at the end of, and 14 to 30 days after camp. We compared the incidence of sputum conversion of patients at camp with that of patients outside camp by culturing specimens from noncamper control subjects with cystic fibrosis who were known not to be infected < or = 2 weeks before and 4 to 6 weeks after camp. We also determined the risk factors for P. cepacia acquisition by determining the relative risk of acquisition between campers who were exposed versus campers who were not exposed to campers known to be infected or to potential environmental sources of P. cepacia at camp. The ribotype of P. cepacia isolates from campers with sputum conversion was compared with that of isolates from other campers and from an environmental source. The cumulative incidence of sputum conversion during the study period was 6.1% (11/181) among campers compared with no incidence (0/92) among noncampers (p = 0.02, Fisher Exact Test). The incidence of sputum conversion at camp varied according to the prevalence of campers with known infection (p < 0.001, chi-square test for trend). The rate of sputum conversion was higher in the camp with longer duration (relative risk = 12.0; 95% confidence interval = 2.7 to 53.5). Ribotyping showed that P. cepacia isolates from all 11 campers with sputum conversion were identical or similar (1 to 2 band difference) to isolates of other P. cepacia-infected campers including co-converters. These results suggest that P. cepacia can be acquired by patients with cystic fibrosis who are attending summer camp for such patients, possibly through person-to-person transmission, and that the risk increases with the prevalence of P. cepacia-infected campers and the duration of camp.

Adolescent↗
PubMed1993

Elevated cAMP gives short-term inhibition and long-term stimulation of hepatocyte DNA replication: roles of the cAMP-dependent protein kinase subunits.

The study reports the role of the isozyme forms (cA-PKI and cA-PKII) and subunits (R and C) of cAMP-dependent protein kinase in mediating the acute depression of hepatocyte DNA replication by elevated cAMP. Combinations of cAMP analogs preferentially activating cA-PKI or II showed that either isozyme could inhibit DNA replication. The effects of glucagon and cAMP analogs were counteracted by the cAMP antagonist RpcAMPS, implicating the necessity for cA-PK dissociation in cAMP action. The effect of elevated cAMP was mimicked by microinjected C subunit, but not by the RI subunit of cA-PK. Hepatocytes under continuous cAMP challenge more than regained their replicative activity. This tardive stimulatory effect of cAMP was enhanced by insulin and blocked by dexamethasone, and was preceded by downregulation of cA-PK. In conclusion, a burst of cAMP acutely inhibits hepatocyte G1/S transition in late G1 regardless of hormonal state. In the presence of high glucocorticoid/low insulin the inhibition persists. At high insulin/low glucocorticoid the inhibitory phase is followed by a prolonged stimulation of DNA replication. Downregulation of endogenous cA-PK is a mechanism for escape from the inhibitory action of highly elevated cAMP.

Animals↗
PubMed2002

A new nonhydrolyzable reactive cAMP analog, (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase.

Levels of cAMP that control critical platelet functions are regulated by cGMP-inhibited cAMP phosphodiesterase (PDE3A). We previously showed that millimolar concentrations of the hydrolyzable 8-[(4-bromo-2,3-dioxobutyl)thioadenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP) inactivate PDE3A. We have now synthesized a nonhydrolyzable affinity label to probe the active site of PDE3A. The nonhydrolyzable adenosine 3',5'-cyclic monophosphorothioates, Sp-cAMPS and Rp-cAMPS, function as competitive inhibitors of PDE3A with K(i) = 47.6 and 4400 microM, respectively. We therefore coupled Sp-cAMPS with 1,4-dibromobutanedione to yield (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, [Sp-cAMPS-(BDB)]. Sp-cAMPS-(BDB) inactivates PDE3A in a time-dependent, irreversible reaction with k(max) = 0.0116 min(-1) and K(I) = 10.1 microM. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) in microM) is: Sp-cAMPS (24) > Rp-cGMPS (1360), Sp-cGMPS (1460) > GMP (4250), AMP (10600), Rp-cAMPS (22170). These results suggest that the inactivation of PDE3A by Sp-cAMPS-(BDB) is a consequence of reaction at the overlap of the cAMP and cGMP binding regions in the active site.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Down regulation of cAMP production by cAMP receptor protein in Escherichia coli: an assessment of the contributions of transcriptional and posttranscriptional control of adenylate cyclase.

Escherichia coli cells that are deficient in the cAMP receptor protein (CRP) overproduce cAMP. We and others have previously found that transcription of the adenylate cyclase gene (cya) is negatively regulated by the CRP-cAMP complex. Here, we have investigated the contribution of this transcriptional regulation to the control of cAMP levels. Several variants of the cya gene have been constructed and characterized with respect to their expression and their ability to produce cAMP. Overproduction of cAMP in a crp- background was reduced from 200-fold to 50-fold when transcriptional regulation by CRP-cAMP was eliminated by replacing the cya promoter with the constitutive bla promoter. When the C-terminal 48 amino acids of adenylate cyclase were deleted without changing the promoter, the degree of overproduction of cAMP was reduced to 4-fold. Finally, the increase in cAMP level observed in crp- cells was almost completely abolished when the truncated cyclase was expressed from the bla promoter. We conclude that transcriptional regulation of cya does indeed play a role in the down-regulation of cAMP production by CRP, although the major regulation is exerted at the posttranscriptional level. The C-terminal region comprising the last 48 amino acids of cyclase is responsible for the posttranscriptional regulation. A simple new method for the determination of cAMP is also described.

Adenylyl Cyclases↗
PubMed1989

Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production.

Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Cell selective cAMP induction of rat CYP1B1 in adrenal and testis cells. Identification of a novel cAMP-responsive far upstream enhancer and a second Ah receptor-dependent mechanism.

CYP1B1 is unique among P450 cytochromes in exhibiting inductive responses mediated by both the Ah receptor (AhR) and cAMP. cAMP induction was mediated either by a 189bp far upstream enhancer region (FUER, -5110 to -5298) or by a 230bp AhR-responsive enhancer region (AhER) (-797 to -1026). CYP1B1 luciferase reporters respond selectively to cAMP and TCDD in adrenal Y-1 cells (only cAMP), testis MA10 cells (cAMP>TCDD), and C3H10T1/2 mouse embryo fibroblasts (only TCDD). In Y-1 cells, which lack AhR, cAMP induction is totally dependent on the FUER, including absolute requirements for upstream and downstream halves of this region, and for CREB activity at a CRE sequence located at the 3(')-end. cAMP stimulation of the FUER was remarkably high (27-fold) and equally effective when linked to an HSV-TK promoter, indicating direct cAMP activation of the FUER. Binding of CREB to the essential CRE was demonstrated along with dominant negative effects of functionally impaired mutants. cAMP induction in MA10 cells was partially mediated by the FUER mechanism but was regulated additionally by AhER through AhR activity. MA10 cells also exhibit cAMP-dependent AhR down-regulation and AhR/Arnt complex formation. Mutations in AhER including XRE5 were similarly inhibitory to cAMP stimulation in MA10 cells and to TCDD stimulation in C3H10T1/2 cells. Transfection of AhR into the AhR-deficient Y-1 cells did not introduce this second mechanism, which indicated a need for additional components that are present in MA10 cells.

Adrenal Glands↗
PubMed1997

Binding of two fluorescent cAMP analogues to type I and II regulatory subunits of cAMP-dependent protein kinases.

Binding of two long wavelength fluorescent cAMP analogues, 8-thioacetamido-fluorescein-cAMP (SAF-cAMP) and 8-thioacetamido-rhodamine-cAMP (SAR-cAMP), to the RI (from bovine muscle) and RII (from bovine heart) regulatory subunits of cAMP dependent kinases has been studied. Displacement of [3H]cAMP from RI and RII and equilibrium dialysis measurements show that the fluorescent nucleotides are high affinity ligands for the cAMP binding sites. The binding is characterized by complex fluorescence spectral and fluorescence anisotropy changes, more evident for the fluorescein than for the rhodamine derivative. The fluorescence excitation spectrum of the bound SAF-cAMP is characterized by the appearance of a red shifted shoulder at 500-510 nm excitation wavelength region. Any change of the bound/free ratio in a solution equilibrium is accompanied by changes in fluorescence and anisotropy signals which are best detected at suitable wavelengths. It is proposed that fluorescence and anisotropy changes can distinguish between binding to type B (slow dissociating) and A (fast dissociating) cAMP binding sites of regulatory subunits. Applications of the fluorescent nucleotides to kinase localization and cAMP determination in living cells are discussed.

Animals↗
PubMed2003

The expression of the thyroid-stimulating hormone (TSH) receptor and the cAMP-dependent protein kinase RII beta regulatory subunit confers TSH-cAMP-dependent growth to mouse fibroblasts.

TSH activates its specific receptor in thyroid cells and induces cAMP, a robust stimulator of thyroid cell proliferation. Conversely, cAMP is a potent inhibitor of growth in mouse fibroblasts. To dissect the signals mediating cAMP-dependent growth, we have expressed in mouse fibroblasts the human thyrotropin receptor (TSHR) or a constitutively active mutant, under the control of the tetracyclin promoter. Both TSHR and cAMP levels were modulated by tetracyclin. In the presence of serum, activation of TSHR by TSH induced growth arrest. In the absence of serum, cells expressing TSHR stimulated with TSH, replicated their DNA, but underwent apoptosis. Co-expression of cAMP-dependent protein kinase (PKA) regulatory subunit type II (RIIbeta) inhibited apoptosis and stimulated the growth of cells only in the presence of TSH. Expression of RIIbeta-PKA, in the absence of TSHR, induced apoptosis, which was reversed by cAMP. Growth, stimulated by TSHR-RIIbeta-PKA in mouse fibroblasts, was also dependent on Rap1 activity, indicating cAMP-dependent growth in thyroid cells. As for the molecular mechanism underlying these effects, we found that in normal fibroblasts, TSH induced AKT and ERK1/2 only in cells expressing TSHR and RII. Similarly, activation of TSHR increased cAMP levels greatly, but was unable to stimulate CREB phosphorylation and transcription of cAMP-induced genes in the absence of RII. These data provide a simple explanation for the anti-proliferative and proliferative effects of cAMP in different cell types and indicate that RII-PKAII complements TSHR action by stably propagating robust cAMP signals in cell compartments.

Adenoviridae↗
PubMed2004

Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype.

cAMP can be either mitogenic or anti-mitogenic, depending on the cell type. We demonstrated previously that cAMP inhibited the proliferation of normal renal epithelial cells and stimulated the proliferation of cells derived from the cysts of polycystic kidney disease (PKD) patients. The protein products of the genes causing PKD, polycystin-1 and polycystin-2, are thought to regulate intracellular calcium levels, suggesting that abnormal polycystin function may affect calcium signaling and thus cause a switch to the cAMP growth-stimulated phenotype. To test this hypothesis, we disrupted intracellular calcium mobilization by treating immortalized mouse M-1 collecting duct cells and primary cultures of human kidney epithelial cells with calcium channel blockers and by lowering extracellular calcium with EGTA. Calcium restriction for 3-5 h converted both cell types from a normal cAMP growth-inhibited phenotype to an abnormal cAMP growth-stimulated phenotype, characteristic of PKD. In M-1 cells, we showed that calcium restriction was associated with an elevation in B-Raf protein levels and cAMP-stimulated, Ras-dependent activation of B-Raf and ERK. Moreover, the activity of Akt, a negative regulator of B-Raf, was decreased by calcium restriction. Inhibition of Akt or phosphatidylinositol 3-kinase also allowed cAMP-dependent activation of B-Raf and ERK in normal calcium. These results suggest that calcium restriction causes an inhibition of the phosphatidylinositol 3-kinase/Akt pathway, which relieves the inhibition of B-Raf to allow the cAMP growth-stimulated phenotypic switch. Finally, M-1 cells stably overexpressing an inducible polycystin-1 C-terminal cytosolic tail construct were shown to exhibit a cAMP growth-stimulated phenotype involving B-Raf and ERK activation, which was reversed by the calcium ionophore A23187. We conclude that disruption of calcium mobilization in cells that are normally growth-inhibited by cAMP can derepress the B-Raf/ERK pathway, thus converting these cells to a phenotype that is growth-stimulated by cAMP.

Animals↗
PubMed1980

Cyclic 3',5'-AMP relay in Dictyostelium discoideum IV. Recovery of the cAMP signaling response after adaptation to cAMP.

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP. Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus. We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.

Adaptation, Physiological↗
PubMed1994

cAMP concentrations, cAMP dependent protein kinase activity, and phospholamban in non-failing and failing myocardium.

OBJECTIVE: Several signal transduction defects such as a reduction of myocardial cAMP formation and an altered intracellular Ca2+ handling have been observed in the failing human myocardium. The aim of the study was to obtain data on changes beyond cAMP formation involving cAMP dependent protein kinase and its substrates. METHODS: cAMP dependent protein kinase activity and cAMP concentrations were measured in the particulate and soluble fraction of failing human hearts (ischaemic, and dilated cardiomyopathy) and non-failing donor hearts. Phospholamban was quantified by cAMP dependent phosphorylation using 32P-ATP as substrate and on western blots using a monoclonal antibody. RESULTS: cAMP concentrations were reduced in the particulate fraction in both ischaemic and dilated cardiomyopathy and in the soluble fraction in dilated cardiomyopathy, but there was no difference in cAMP dependent protein kinase activity. Both phospholamban levels and cAMP dependent phosphorylation of phospholamban were similar in non-failing myocardium and in both ischaemic and dilated cardiomyopathy. CONCLUSIONS: These findings show that the reduction of cAMP formation is the predominant alteration in heart failure, but cAMP dependent protein kinase and phospholamban are evidently unchanged.

Adenosine Triphosphatases↗
PubMed1990

Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of the E. coli cAMP receptor protein.

Wild type cAMP receptor protein (CRP) activates in vitro lac transcription only in the presence of cAMP. In contrast the mutant CRP*598 (Arg-142 to His, Ala-144 to Thr) can activate lac transcription in the absence of cyclic nucleotide or at concentrations of cAMP below that required by CRP. To further characterize the properties of CRP*598, the binding of cAMP and cGMP to CRP and CRP*598 has been determined. The intrinsic binding constant (K) values obtained for cAMP binding are: CRP, 1.9 x 10(4) M-1; CRP*598, 3.8 x 10(5) M-1. The K values obtained for cGMP binding are: CRP, 2.9 x 10(4) M-1; CRP*598, 2.7 x 10(4) M-1. The results indicate that the affinity of CRP and CRP*598 for cGMP is relatively unchanged while the affinity of CRP*598 for cAMP is approximately twenty times greater than that shown by CRP. Binding of cAMP by CRP and cGMP by CRP or CRP*598 exhibits slight negative cooperativity. The major difference seen is that CRP*598 binds cAMP with strong positive cooperativity. The importance of the unsubstituted N6 position of the adenine moiety is also shown by the similar affinity of both forms of CRP for N6-butyryl cAMP. The cAMP binding properties evinced by CRP*598 suggest that its intrinsically altered conformation may be related to that assumed by CRP in a CRP-DNA or a cAMP-CRP-DNA complex.

Adenosine↗
PubMed1994

Fibroblast growth factor and cyclic AMP (cAMP) synergistically activate gene expression at a cAMP response element.

Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways.

Activating Transcription Factor 2↗
PubMed2005

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP.

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K(+), ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca(2+)) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca(2+) content are controversial and far from being completely resolved. Here, we found that Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2'-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca(2+) or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.

Adrenocorticotropic Hormone↗
PubMed2002

Extracellular cAMP inhibits proximal reabsorption: are plasma membrane cAMP receptors involved?

Glucagon binding to hepatocytes has been known for a long time to not only stimulate intracellular cAMP accumulation but also, intriguingly, induce a significant release of liver-borne cAMP in the blood. Recent experiments have shown that the well-documented but ill-understood natriuretic and phosphaturic actions of glucagon are actually mediated by this extracellular cAMP, which inhibits the reabsorption of sodium and phosphate in the renal proximal tubule. The existence of this "pancreato-hepatorenal cascade" indicates that proximal tubular reabsorption is permanently influenced by extracellular cAMP, the concentration of which is most probably largely dependent on the insulin-to-glucagon ratio. The possibility that renal cAMP receptors may be involved in this process is supported by the fact that cAMP has been shown to bind to brush-border membrane vesicles. In other cell types (i.e., adipocytes, erythrocytes, glial cells, cardiomyocytes), cAMP eggress and/or cAMP binding have also been shown to occur, suggesting additional paracrine effects of this nucleotide. Although not yet identified in mammals, cAMP receptors (cARs) are already well characterized in lower eukaryotes. The amoeba Dictyostelium discoideum expresses four different cARs during its development into a multicellular organism. cARs belong to the superfamily of seven transmembrane domain G protein-coupled receptors and exhibit a modest homology with the secretin receptor family (which includes PTH receptors). However, the existence of specific cAMP receptors in mammals remains to be demonstrated. Disturbances in the pancreato-hepatorenal cascade provide an adequate pathophysiological understanding of several unexplained observations, including the association of hyperinsulinemia and hypertension, the hepatorenal syndrome, and the hyperfiltration of diabetes mellitus. The observations reviewed in this paper show that cAMP should no longer be regarded only as an intracellular second messenger but also as a first messenger responsible for coordinated hepatorenal functions, and possibly for paracrine regulations in several other tissues.

Amino Acid Sequence↗
PubMed1996

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

8-Bromo Cyclic Adenosine Monophosphate↗