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PubMed1979

Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis.

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.

Affinity Labels↗

PubMed1989

[The effect of 3',5'-cyclic adenylic acid (cAMP) on the growth of bacteria].

The effect of intracellular cAMP level on the growth of bacteria was studied with E. coli AS 1.797, Corynebacterium pekinense AS 1.299 and Bacillus megaterium AS 1.217. The experimental results show that the growth of E. coli AS 1.797 was decreased with increasing cAMP level in cell. With glucose as the sole carbon source, intracellular cAMP is low, and growth of E. coli AS 1.797 was inhibited by exogenous cAMP, but the analog metabolite of cAMP-5'-AMP had no effect on growth. When E. coli AS 1.797 was grown in media containing lactose, maltose and glycerin respectively, intracellular cAMP level is high, and exogenous cAMP did not inhibited the cell growth. When Corynebacterium pekinense AS 1.299 took glucose as carbon source the growth of this bacterium was also inhibited by exogenous cAMP, but the inhibition of cAMP was not specific, its role can be exchanged by 5'-AMP. B. megaterium AS 1.217 cultivated in different carbon source (include glucose) do not contain cAMP in cell, and its growth was not inhibited by exogenous cAMP. 5'-AMP had also no effect on the growth of this bacterium. Thus, the conclusion seems that the cAMP is not essential for the growth of bacteria, and it is a regulator that act a negative regulation in cell growth, but this regulator is of no effect for the growth of B. megaterium AS 1.217.

Bacillus megaterium↗

PubMed1989

Alkylated cAMP derivatives: selective synthesis and biological activities.

Many alkylated cAMPs have been prepared and tested for their antitumor and cardiac activities. Treatment of cAMP with several bases and alkyl bromides gave alkyltriesters of cAMP (2), N6,N6,2'-O-trialkyl cAMPs (3), N6,2'-O-dialkyl cAMPs (4) and 2'-O-monoalkyl cAMPs (5) in one step. N6,N6-dialkyl cAMPs (6) were prepared from 2'-O-tosyl cAMP by the similar alkylation, followed by detosylation. Synthesis of N6-monoalkyl cAMPs (7) was achieved by the reductive alkylation of cAMP with aldehydes in one step. Alkyl triesters of cAMP exhibited antitumor activities against P815 cells. N6-mono and N6,N6-dialkyl cAMPs showed significant cardiac activities.

Alkylation↗

PubMed1989

The cyclic nucleotide specificity of eight cAMP-binding proteins in Dictyostelium discoideum is correlated into three groups.

cAMP is a mediator of inter- and intracellular events in Dictyostelium discoideum and is thought to act through specific receptors. Eight forms of cAMP-binding proteins have been described in this organism: four forms of a cell surface receptor, a cell surface and extracellular phosphodiesterase, an intracellular cAMP-dependent protein kinase (CAK), and a recently identified cAMP-binding protein (CABP1) that is present on the cell surface, in the cytoplasm, and in the nucleus. In this study we have analyzed the cyclic nucleotide specificity of these cAMP-binding proteins using 13 derivatives of cAMP with modifications in the adenine, ribose, and phosphate moiety. The results suggest that the cAMP-binding proteins belong to three groups: (i) four forms of the cell surface receptor, (ii) two forms of an intracellular receptor (CABP1 and CAK), and (iii) cell surface and extracellular phosphodiesterase. cAMP is probably bound to the surface receptors in the anti conformation in a hydrophobic cleft of the receptor with essential interactions at N6H2' and O3'. In contrast, cAMP is probably bound to CAK and CABP1 in the syn conformation with essential interactions at O2', O3', O5', and exocyclic oxygen. Finally, binding of cAMP to phosphodiesterase involves only O3' and exocyclic oxygen. The cyclic nucleotide specificity of cAMP-induced processes in D. discoideum indicates that the cell surface receptors participate in the transduction of the cAMP signal during chemotaxis and cell differentiation. Functions for CABP1 and CAK in these processes are presently elusive.

Carrier Proteins↗

PubMed1982

Alterations of glial tumor cell Ca2+ metabolism and Ca2+-dependent cAMP accumulation by phorbol myristate acetate.

C6 glial tumor cells exposed to phorbol myristate acetate (PMA) possessed lowered cAMP content, reduced ability to accumulate cAMP in response to norepinephrine or cholera toxin, and a 3-fold increase in the concentration of norepinephrine producing 50% of the maximal rate of cAMP accumulation. Detectable effects on cAMP accumulation occurred within 10 min of exposure to PMA, and prominent effects by 2 h. PMA similarly affected cells pretreated with cycloheximide. In contrast, Ca2+-depleted preparations of control and PMA-treated cells accumulated cAMP identically in response to norepinephrine or cholera toxin. Ca2+ restoration, which increased the rate of cAMP accumulation in control cells severalfold, did not enhance cAMP accumulation in PMA-treated cells. Neither high catecholamine nor high extracellular Ca2+ concentrations reversed the suppression of cAMP accumulation by PMA. Trifluoperazine, which inhibited the Ca2+-dependent component of norepinephrine-stimulated cAMP accumulation in control cells, did not significantly reduce norepinephrine-stimulated cAMP accumulation in PMA-treated cells. Cell free preparations of control and PMA-treated cultures did not differ significantly in calmodulin content or in Ca2+-stimulated adenylate cyclase, Ca2+-dependent cAMP phosphodiesterase, and (Ca2+-Mg2+)-ATPase activities. The Ca2+ content, however, of intact cells decreased with time of PMA treatment. Within minutes after exposure to PMA, the ability of Ca2+-depleted cells to take up 45Ca was significantly reduced. Both 45Ca uptake and Ca2+-dependent cAMP accumulation were reduced over the same PMA concentration range.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1983

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast.

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.

Chromatography, Ion Exchange↗

PubMed1998

Outward currents in Drosophila larval neurons: dunce lacks a maintained outward current component downregulated by cAMP.

Outward current modulation by cAMP was investigated in wild type (wt) and dunce (dnc) Drosophila larval neurons. dnc is deficient in a cAMP phosphodiesterase and has altered memory. Outward current modulation by cAMP was investigated by acute or chronic exposure to cAMP analogs. The analysis included a scrutiny of outward current modulation by cAMP in neurons from the mushroom bodies (mrb). In Drosophila, the mrb are the centers of olfactory acquisition and retention. Based on outward current patterns, neurons were classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs was reversible and found only in type I and type IV neurons. In the general wt neuron population, approximately half of neurons exhibited cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. On the other hand, a significantly larger fraction of mrb neurons in wt (70%) was endowed with cAMP-modulated, 4-AP-sensitive currents. Only 30% of the dnc neurons displayed outward currents modulated by cAMP. The deficit of cAMP-modulated outward currents was most severe in neurons derived from the mrb of dnc individuals. Only 4% of the mrb neurons of dnc were cAMP-modulated. The dnc defect can be induced by chronic exposure of wt neurons to cAMP analogs. These results document for the first time a well defined electrophysiological neuron phenotype in correlation with the dnc defect. Moreover, this study demonstrates that in dnc mutants such a deficiency affects most severely neurons in brain centers of acquisition and retention.

Animals↗

PubMed1998

The mechanism of 8-Cl-cAMP action.

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is a potential new anticancer agent, but its mechanism of action is not clearly defined. In this work we have studied the effect of various heat inactivated and heat untreated human sera in the absence or in the presence of a nonspecific phosphodiesterase (PDE) inhibitor, IBMX, or of nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole (DP), or of inosine-monophosphatedehydrogenase (IMPDH) inhibitor, tiazofurin, (T), on the antiproliferative 8-Cl-cAMP action towards two human malignant cell lines, K562 and HeLa cells, in vitro. Cell survival was determined 72 hrs after the agents action, using MTT assay. The results obtained, indicated the similar inhibitory effect of 8-Cl-cAMP on HeLa cell survival in the presence of four different heat untreated human sera (IC50 = 4-4.8 microM). Serum heat inactivation caused decrease in 8-Cl-cAMP antiproliferative action depending on the blood donor (IC50 = 23 microM, 15 microM, 19 microM, and 9 microM) and suggesting that some thermolabile ingredient(s) present in sera is involved, at least partially, in the induction or permittance of antiproliferative 8-Cl-cAMP action. K562 Cells were not as much resistant to 8-Cl-cAMP as HeLa cells, or mouse melanoma B16 cells; in the presence of heat untreated FBS, IC50 = 16 microM, while for B16 cells IC50 was 8 microM. Different human sera show different effect on 8-Cl-cAMP action on K562 cells: IC50 = 7.5 microM and 16.5 microM. In the presence of heat inactivated human sera 8-Cl-cAMP IC50 concentrations were higher, with relevant mutual differences. The effect of different sera on 8-Cl-cAMP action was only partly abrogated in the presence of a nonspecific PDE inhibitor, IBMX, suggesting that the serum PDE action is one of the various factors contributing to the induction of 8-Cl-cAMP antiproliferative action. Nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole inhibited the antiproliferative 8-Cl-cAMP action to HeLa and K562 cells. Tiazofurin and 8-Cl-cAMP acted as antagonists on HeLa, but not on K562 cells.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Therapeutic camping for children with end-stage renal disease.

Therapeutic camping experiences for children with end-stage renal disease (ESRD) have proliferated in the United States and abroad. This report is based on the results of a survey designed to accumulate data on the development and implementation of 20 such camps. Children attending camp ranged in age from 1 year to 19 years. Single disease-specific camps were most common, while camps for children with a variety of chronic illnesses, including ESRD, and mainstream camps were also conducted. Facilities were available for hemodialysis and continuous ambulatory peritoneal dialysis, but not automated peritoneal dialysis, in the majority of surveyed camps. Dialysis nurses, pediatric nephrologists, dietitians and social workers were the medical personnel that most frequently participated in the camps. On average, 32 dialysis/transplant patient campers (range 6-100) attended camp for a 1-week session. Therapeutic camping experiences for children with ESRD are extremely successful and attempts to increase the availability of similar camps should be encouraged.

Adolescent↗

PubMed1994

Role of cAMP in the reactivation of demembranated ram spermatozoa.

Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was approximately 100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained > 90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase.

Adenosine Triphosphate↗

PubMed1986

DNA-mediated transfer of cAMP resistance in CHO cells.

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP-dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP-dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.

3T3 Cells↗

PubMed1996

Simulations of CRP:(cAMP)2 in noncrystalline environments show a subunit transition from the open to the closed conformation.

The CRP:cAMP complex functions as a transcription factor that facilitates RNA polymerase recognition of several bacterial promoters. Detailed crystal structure information is available for CRP:(cAMP)2 and for CRP:(cAMP)2 complexed with DNA. In the crystalline environment, CRP:(cAMP)2 subunits are asymmetrically related; one subunit has a closed conformation and the other has an open conformation. The CRP:(cAMP)2 complexed with DNA shows both subunits in a closed conformation. We have studied the molecular dynamics of CRP:(cAMP)2 in noncrystalline environments. CRP:(cAMP)2 was simulated for 625 ps in vacuo and for 140 ps in solution. The crystal structure of CRP:(cAMP)2 in the absence of DNA was used as the initial conformation. Molecule optimal dynamic coordinates (MODCs) (García A, 1992, Phys Rev Lett 68:2696) were used to analyze protein conformations sampled during the course of the simulations. Two MODCs define a transition of the open subunit to a closed subunit conformation during the first 125 ps of simulation in vacuo; the resulting subunit conformation is similar to that observed in CRP:(cAMP)2:DNA crystals. Simulation of CRP:(cAMP)2 in solution showed that a transition from the open to the closed state also occurs when water is explicitly included in the calculations. These calculations suggest that the asymmetric conformation of CRP:(cAMP)2 is stabilized by crystal lattice interactions. The predicted solution conformation is more symmetric, with both subunits in a closed conformation.

Adenosine Monophosphate↗

PubMed1995

Stimulation of avian myoblast differentiation by triiodothyronine: possible involvement of the cAMP pathway.

In a previous work, we have shown that T3 induces a potent stimulation of avian myoblast differentiation. In this study, we demonstrated that this hormone did not affect MyoD and myogenin expression. As numerous data suggest that T3 could affect the cAMP pathway, we have studied its involvement in the myogenic activity of triiodothyronine on quail myoblast. In agreement with Zalin and Montagues (Cell 2, 103-108 (1974)), we observed a transient rise in myoblast intracellular cAMP level some hours before the onset of terminal differentiation. Interestingly, this rise occurred earlier in T3-treated than in control myoblasts, and cAMP production was significantly increased by the hormone. Moreover, T3 increased CREB transcriptional activity, thus suggesting that the entire cAMP signaling pathway was stimulated by this hormone. In addition, we observed that addition of an inhibitor of adenylate cyclase activity prior to the cAMP rise dramatically inhibited myoblast differentiation. Last, we showed that cAMP mimicked all T3 actions upon myoblast differentiation: (1) T3 and cAMP reduced myoblast proliferation by increasing the number of postmitotic myoblasts at cell confluence; (2) T3 and cAMP increased BTG1 nuclear accumulation; (3) T3 and cAMP stimulated terminal differentiation only when added during the proliferative phasis. These data strongly suggest that the transient rise in cAMP production could be essential for myoblast terminal differentiation. In addition, it appears that, at least in avian myoblasts, T3 stimulation of terminal differentiation involves the cAMP pathway.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1.

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.

Binding Sites↗

PubMed1992

cAMP activation of CF-affected Cl- conductance in both cell membranes of an absorptive epithelium.

Cystic fibrosis (CF) is characterized by abnormal epithelial Cl- conductance (GCl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected GCl(CF-GCl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though GCl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-GCl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-GCl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: beta-adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased Gt by about 2.3-fold (n = no. of ducts = 8). Removal of media Cl-, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of GCl. cAMP activated both apical and basolateral GCl. cAMP hyperpolarized gluconate: Cl- (lumen:bath) transepithelial bionic potentials (delta Vt = -20.3 +/- 5.2 mV, mean +/- SE, n = 9) and transepithelial 3: 1 luminal NaCl dilution diffusion potentials (delta Vt = -8.8 +/- 2.9 mV, n = 5). cAMP activated basolateral GCl as indicated by increased bi-ionic (gluconate:Cl-, bath:lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (Vt = -6.9 +/- 0.8 mV, n = 24, nonresponsive vs. -19.4 +/- 1.8 mV, n = 22, responsive ducts) but larger bi-ionic potentials (-94 +/- 6 mV, n = 35, nonresponsive vs. -65 +/- 5 mV, n = 17, responsive ducts) and dilution diffusion potentials (-40 +/- 5 mV, n = 11, nonresponsive vs. -29 +/- 3 mV, n = 7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of GCl in nonresponsive ducts and submaximal activation of GCl in responsive ducts. We conclude that cAMP activates CF-GCl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.

Adrenergic beta-Agonists↗

PubMed1990

Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins.

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

2,4-Dinitrophenol↗

PubMed1982

Mechanism of adenosine 3',5'-monophosphate (cAMP)-induced increase in Ca2+ uptake by the sarcoplasmic reticulum in functionally skinned myocardial fibers.

The increased rate of Ca2+ uptake and ATPase activity in isolated cardiac sarcoplasmic reticulum (SR) by adenosine 3',5'-monophosphate (cAMP) has been shown to be activated by a cAMP-dependent protein kinase (cAMP kinase). Functionally skinned myocardial fiber preparations were used to study the mechanisms of cAMP action on the SR at the same time that tension was monitored. cAMP effects were studied on Ca2+ -activated tension of the contractile proteins, and on Ca2+ uptake and release from the SR using caffeine-induced tension transients. Neither cyclic AMP (0.1-5 microM) nor the catalytic subunit of cAMP kinase (0.1-1 microM) (PK-C) significantly changed either the maximal or the submaximal Ca2+ -activated tension. The areas of the tension transients were unchanged when cAMP was present in the releasing solution (release phase), and were significantly increased up to a mean of about 80% when cAMP or PK-C was present in the Ca2+ loading solutions (uptake phase). The increased tension transient was blocked by heat-stable inhibitor of cAMP kinase. We conclude that cAMP-induced increases in Ca2+ uptake by the SR could play an important role in the positive inotropic effect. cAMP kinase could thus play a crucial role in the regulation of myocardial contractility.

Animals↗