Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene (original) (raw)
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Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells
European Journal of Biochemistry, 1990
In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 1251-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the CAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1 -5 mM, intracellular and extracellular (medium) CAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 FM) elicited a CAMP response similar to that found with the toxin, and a similar inhibition of CAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal CAMP levels which were reduced by 40-60% in cells cultured for 24-48 h with the fatty acid. In order to study whether butyrate influenced CAMP synthesis andlor CAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40 -50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40 -50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells.
Short-term feedback regulation of cAMP by accelerated degradation in rat tissues
Journal of Biological Chemistry
A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes (Corbin, J. D., Beebe, S . J., and Blackmore, P. F. (1985) J. Biol. Chern. 260, 8731-8735). The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the CAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pC14S-cAMP (where 8-pClI#JS-indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in CAMP. More than 20% of the cAMP binding sites on the protein kinase were unoccupied at concentrations of 8-pClgS-cAMP that produced maximal cAMP lowering. Thus, the possibility that 8-pCl4S-cAMP lowered cAMP by displacing it from protein kinase binding sites, making it available for hydrolysis, seemed unlikely. In adipocytes, the lowering of cAMP by 8-pC14S-cAMP occurred in parallel with increases in lipolysis and activation of low K,,, phosphodiesterase, suggesting that the phosphodiesterase was responsible for the cAMP lowering. Further evidence for this assertion was the finding that in hepatocytes preloaded with low concentrations of 8-pCL$S-cAMP, glucagon lowered 8-pClI#JS-cAMP by about 50%, an amount similar to the cAMP lowering observed with 8-pClI#JS-cAMP treatment. The results were consistent with a CAMP-dependent protein kinase-catalyzed activation of a phosphodiesterase and suggested that 8-pCl4S-CAMP-mediated hydrolysis of cAMP mimicked a physiologically significant response. The observation of this phenomenon in several tissues further suggested that it may be a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of CAMP.
European Journal of Pharmacology: Molecular Pharmacology, 1994
Stable Sl15 mouse mammary tumour cell lines, expressing separately a2A-C10, a2B-C2 and a2C-C4 adrenoceptors were used to compare the receptor binding properties of a2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 /zM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the a2A-C10 subtype, smaller in a2C-C4, and minimal in a2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the a2B-C2 subtype (35%) than in the aeA-C10 and a2C-C4 subtypes (50-70%). After treatment of cells expressing a2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by a2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) a2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected Sl15 cells, (2) all three a2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in Sl15 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in Sl15 cells is different among the subtypes so that the a2A-C10 subtype is coupled with high efficacy but with low sensitivity, the a2B-C2 subtype with low efficacy but high sensitivity, and the a2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least aeB-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through G s.