Heterologous desensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells.
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ABSTRACT:
SUBMITTER: Breant B
PROVIDER: S-EPMC1163571 | biostudies-other | 1981 Dec
REPOSITORIES: biostudies-other
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Project description:Vasopressin and alpha-adrenergic agonists were shown previously [Bréant, Keppens & De Wulf (1981) Biochem. J. 200, 509-514] to induce a heterologous, dose-dependent and receptor-mediated desensitization of the cyclic AMP-independent glycogenolytic response in isolated hepatocytes. The desensitized state of the hepatocytes can be preserved as long as the agonist is bound to its receptor. Conversely, washing the cells with a hormone-free buffer or displacement of the agonist from its receptor by a specific antagonist restores the responsiveness. The desensitization and its reversibility (i.e. resensitization) are obtained within minutes. The desensitization can be clearly elicited at temperatures as low as 5 degrees C, whereas the glycogenolytic response and the enhancement of the 45Ca flux are only obtained above 15 degrees C; the resensitization requires even higher temperatures. A tentative model is proposed to account for the observed effects.
| S-EPMC1153966 | biostudies-other
Project description:We have studied the effect of pig pancreastatin on glucose and lactate production in freshly isolated rat hepatocytes. Pancreastatin stimulated the rate of glucose output, whereas, in contrast with glucagon, it failed to modify the rate of lactate production. The effective concentration of pancreastatin was in the range 0.1-100 nM, with half-maximal rate close to 1 nM. The ability of pancreastatin to increase glucose output was abolished by chelation of the calcium in the medium. By itself, pancreastatin did not increase cyclic AMP (cAMP) levels and had no influence on cAMP levels in glucagon-stimulated hepatocytes. Our results point out a possible role of pancreastatin in glycogenolysis. This appears to be mediated by a cAMP-independent Ca(2+)-dependent mechanism.
| S-EPMC1132588 | biostudies-other
Project description:Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.
| S-EPMC4556939 | biostudies-literature
Project description:Addition of 1-3 mM-8-bromo-cyclic AMP to monolayer cultures of H-4 rat hepatoma cells resulted in a rapid but short-lived increase in tyrosine aminotransferase (EC 2.6.1.5) activity. The transient nature of this induction is due to desensitization to 8-bromo-cyclic AMP. Throughout this time course of induction and desensitization, removal of 8-bromo-cyclic AMP resulted in a rapid and significant decrease in tyrosine aminotransferase activity, a process referred to as 'de-induction' in this study. We showed that the changes in tyrosine aminotransferase activity in its induction, desensitization and de-induction by 8-bromo-cyclic AMP were directly attributable to changes in the synthesis rate of the protein, and the amount of translatable and hybridizable mRNA encoding for tyrosine aminotransferase (mRNATAT). We further showed that this desensitization was specific to cyclic AMP. First, only active analogues of cyclic AMP and agents which increased cellular concentrations of cyclic AMP elicited this desensitization. Second, the desensitized cells were refractory only to the effects of 8-bromo-cyclic AMP; dexamethasone and insulin induced the tyrosine aminotransferase activity in the 8-bromo-cyclic AMP-desensitized cells in a manner similar to that of the controls. Studies on the metabolism of 8-bromo-cyclic AMP suggest that neither its degradation nor the accumulation of its primary metabolite, 8-bromoadenosine, played a significant role in modulating the expression of tyrosine aminotransferase during the time course of action of 8-bromo-cyclic AMP. These results provide evidence for a specific pretranslational mode of action of cyclic AMP in the control of tyrosine aminotransferase expression in its desensitization and de-induction, in addition to the early phase of induction.
| S-EPMC1148992 | biostudies-other
Project description:FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds-lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB-pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.IMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements-including the PilSR two-component system-to cell poles for optimal function.
| S-EPMC5527385 | biostudies-literature
Project description:Study the effect of forskolin, dipyridamol and IBMX (here called FID) supplementation in the in vitro maturation medium for an incubation of 6 hrs on the transcriptomics of bovine cumulus cells.
2015-07-01 | GSE67968 | GEO
Project description:We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.
| S-EPMC1152138 | biostudies-other
Project description:1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.
| S-EPMC1565491 | biostudies-literature
Project description:The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.
| S-EPMC1152604 | biostudies-other
Project description:Study the effect of forskolin, dipyridamol and IBMX (here called FID) supplementation in the in vitro maturation medium for an incubation of 6 hrs on the transcriptomics of bovine cumulus cells. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) supplemented with FID (CC-FID) vs. cumulus cells without FID (Ct-6H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
2015-07-01 | E-GEOD-67968 | biostudies-arrayexpress