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R W Butcher

Publications and source records attributed to R W Butcher.

At least 19 recordsLinked to original sources
PubMed1992

Beta-adrenergic receptor levels and function after growth of S49 lymphoma cells in low concentrations of epinephrine.

Growth of S49 wild-type (WT) lymphoma cells for 24 hr with 3 nM epinephrine produces a very pronounced attenuation of cAMP accumulation in response to subsequent challenges with much higher concentrations of the catecholamine [Mol. Pharmacol. 36:459-464 (1989)]. We report here the effects of this treatment, in S49 WT, cyc-, and kin- cells, on the responsiveness of adenylate cyclase in partially purified membranes. The desensitization of adenylate cyclase in the S49 WT cells after 24-hr treatment was homologous, in that only responses to epinephrine were attenuated. The EC50 for epinephrine stimulation of adenylate cyclase was 54 +/- 8% (mean +/- standard error) higher in treated cells than in controls, and there was a 32 +/- 3% reduction in Vmax at supramaximal epinephrine concentrations. The treatment also caused a 34 +/- 9% reduction in the levels of the beta-adrenergic receptor (beta AR), which was of a sufficient magnitude to account for the homologous desensitization seen. The 24-hr treatment had similar effects in S49 kin- cells, where we observed a 28 +/- 4% decrease in Vmax, a 35 +/- 6% increase in EC50 for epinephrine stimulation of adenylate cyclase, and a 25 +/- 3% decrease in beta AR. In contrast, the 24-hr treatment had no measurable effect on adenylate cyclase activity in S49 cyc- cells. That is, the responsivity of adenylate cyclase reconstituted with Gs from S49 WT cells was not attenuated, although beta AR levels were significantly decreased. The desensitization of S49 cells with the 24-hr treatment was additive with that mediated by the cAMP-dependent protein kinase (cAPK). Further, unlike the cAPK-mediated attenuation, it was relatively insensitive to the levels of free Mg2+ in the adenylate cyclase reaction mixture. The characteristics of the desensitization produced by 24-hr treatment with 3 nM epinephrine, together with the observation that it is similar in S49 WT and kin- cells, demonstrates that the process in WT cells is, at least in part, independent of the rapid cAPK-mediated desensitization. It is also most likely that it is unrelated to the rapid cAMP-independent processes involving sequestration/internalization or the beta AR kinase, because those mechanisms require much higher receptor occupancies than the 0.2% occurring with 3 nM epinephrine. Thus, 24-hr treatment appears to produce attenuation of adenylate cyclase by causing down-regulation of beta AR, without involving any other known form of desensitization.

Adenylyl Cyclases↗
PubMed1992

Positively cooperative cAMP phosphodiesterase attenuates cellular cAMP responses.

We have shown that growth of S49 WT mouse lymphoma cells for 24 hr in 3 nM epinephrine produced very significant desensitization of subsequent cellular cAMP responses to challenges with higher concentrations of epinephrine. The effects of this long-term treatment (LTT) were obvious in intact cells and also when adenylate cyclase activity was measured in semi-purified membranes. Beta 2-adrenergic receptors (beta 2AR) were decreased by LTT, and the desensitization of adenylate cyclase was due at least in part to this down-regulation. When mouse L cells transfected with WT beta 2AR from hamster lung (L-WT beta 2AR cells) were subjected to LTT, the attenuation of adenylate cyclase in membranes was obvious, but the consequences of LTT on intact L-WT beta 2AR cells were highly equivocal. That is, when the effects of epinephrine on cellular cAMP levels were measured in LTT or control L-WT beta 2AR cells little desensitization was apparent. Further, cellular cAMP excursions in response to even very high concentrations of epinephrine were very small in control L-WT beta 2AR cells as compared to control S49 WT cells. Subsequent experiments have shown that L-WT beta 2AR cells possess a phosphodiesterase (PDE) which demonstrates marked positive cooperativity with cAMP with a Hill coefficient of 2. The EC50 for cAMP hydrolysis was approximately 30 nM in cell free preparations. cGMP was a positive allosteric effector of the L-WT beta 2AR cell PDE. Further, when cellular cAMP levels in intact L-WT beta 2AR cells were raised above a threshold by treatment with 0.5 microM forskolin and 2 mM IBMX with the epinephrine challenge, the effect of LLT became obvious in the intact cell system. These data demonstrate that cAMP responses to hormones are greatly decreased in systems where the predominant PDE demonstrates positive cooperativity for cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1990

cAMP-dependent protein kinase responses of S49 cells are reduced by growth in low epinephrine concentrations.

S49 wild-type mouse lymphoma cells grown in 3 nM epinephrine are extensively desensitized. Cellular cAMP responses to subsequent challenges with 100 nM epinephrine are reduced by as much as 80-90%. In this report, we document that protein kinase activity ratios were also attenuated. For example, the activity ratios in naive cells were increased from 0.26 +/- 0.02 to 0.72 +/- 0.04 by incubation with 100 nM epinephrine for 2 min, whereas in cells grown in 3 nM epinephrine for 24 hr before the experiment they were 0.19 +/- 0.02 and 0.29 +/- 0.03. Attenuated protein kinase activity ratios were obvious at epinephrine challenge concentrations ranging from 10 to 1000 nM. Three kinds of experiments provided evidence that the reduced ratios in desensitized cells were secondary to diminished cAMP responses rather than to changes in the cAMP-dependent protein kinase itself. Firstly, when protein kinase activity ratios were plotted against cAMP levels in naive and desensitized cells, the points fell along a common line. Secondly, cell-free cAMP-dependent protein kinase preparations from naive or epinephrine-treated cells had similar activities in the presence of maximal exogenous cAMP and similar half-maximal cAMP concentrations. Finally, the levels of cAMP-binding proteins in extracts from naive and desensitized cells were essentially identical. We conclude that desensitization of S49 cells by very low levels of epinephrine significantly reduced cAMP-dependent protein kinase responses to much higher concentrations of the catecholamine.

Animals↗
PubMed1989

Growth of S49 cells in low concentrations of beta-adrenergic agonists causes desensitization.

Epinephrine at concentrations approximating circulating levels in resting subjects produced significant desensitization in wild type S49 lymphoma cells after long term treatment. Desensitization by such low levels of catecholamines was measured by examining subsequent responses of the cells to higher agonist concentrations and was quantified by comparing the integral cAMP accumulations with time in naive and epinephrine-treated cells challenged with the higher epinephrine concentrations. The cells were significantly desensitized after 8 hr of treatment with 3 nM epinephrine or 3 nM terbutaline and were essentially maximally refractory after 24 hr. The 3 nM epinephrine treatment resulted in a small right shift of the EC50. Responses to epinephrine were partially restored by incubating desensitized cells for 8 hr or longer in growth medium that was free of epinephrine. The attenuation of cAMP responses was largely specific, in that the decrease in the response to prostaglandin was small and the response to forskolin was unchanged. This, together with small increases in cAMP destruction in cell-free preparations from treated cells, suggested that higher phosphodiesterase activity contributed in a minor way to the desensitization. However, the response of the adenylate cyclase system to epinephrine was dramatically attenuated, and very significant changes in the properties of the beta-adrenergic receptors were also obvious. That is, the number of binding sites for epinephrine was reduced by about 65% while the number of sites for [125I]iodocyanopindolol was unchanged. The affinity for the radioactive ligand was significantly reduced. Wild type S49 cells remained viable after several days of continuous treatment with 3 nM epinephrine or terbutaline but responded to subsequent increases in cellular cAMP levels with the expected growth arrest and cytolysis. Involvement of cAMP-dependent protein kinase in this type of desensitization was suggested by the observation that S49 kincells were not desensitized by long term incubation with 3 nM epinephrine. Further, low concentrations of dibutyryl cAMP mimicked the effect of low level epinephrine treatment. We conclude that circulating levels of epinephrine in intact animals are sufficiently high to cause desensitization in cells with sensitivities to the catecholamines in the same range as that of the S49 lymphoma cell in vitro. We would predict that cells with those characteristics would always be at least partially desensitized in vivo.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Direct effects of the pyrimido-pyrimidine derivative RA 233 (Rapenton) on rat 13762NF mammary tumor cell clones in vitro.

Studies with the pyrimido-pyrimidine analogue RA 233 (Rapenton) suggest that its antimetastatic action may not be mediated entirely by inhibition of platelet function. Little is known about its direct effects on tumor cells. We investigated the in vitro effects of RA 233 on clones MTLn3 and MTC of differing metastatic potentials, isolated from the 13762NF rat mammary adenocarcinoma. The results indicated that RA 233 is cytostatic (EC50 of approximately 140 microM and approximately 180 microM for MTLn3 and MTC cells, respectively) rather than cytotoxic by determining changes in viable cell number, thymidine uptake, and incorporation of thymidine and methionine. In both clones RA 233 inhibited cAMP-dependent phosphodiesterase activity and affected cAMP accumulation in intact cells. In contrast, clonal heterogeneity in drug-induced morphological changes, such as vacuole formation and altered organization of cytoskeletal structures, as well as increased tumor cell growth at 50 microM RA 233 was observed between clones MTLn3 and MTC. These data could explain the conflicting results obtained with RA 233 when evaluated as an antimetastatic agent.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Role of high affinity cAMP phosphodiesterase activities in the response of S49 cells to agonists.

Quantitative analysis of a drug or hormone action expressed through increased rates of cAMP synthesis is dependent on the activities and affinities of the phosphodiesterases (PDEs) that hydrolyze the cAMP. It is shown herein that, on both theoretical and experimental grounds, significant contribution to hydrolysis by high affinity PDE activity would lead to striking departures from proportionality of the relationships between rates of cAMP synthesis and intracellular cAMP accumulations. Simulations of cAMP decay curves, cellular concentration-response curves to agonists, and cAMP time courses were used to predict the consequences of a high affinity PDE in an intact cell experimental system. Specifically, the simulations predicted 1) an upward convexity in the cAMP log-decay curve, 2) an upward concavity in the concentration-response curve, and 3) the amplification of small differences in initial rates resulting in large differences in accumulation at long times. S49 WT lymphoma cells demonstrated properties that fitted the predictions of the simulations in an experimental system. We conclude that the presence of significant high affinity PDEs has a profound effect on the nature of the response of cells to agonists and antagonists of the adenylate cyclase system. In addition, intracellular rates of hydrolysis were compared with PDE activities measured in cell-free systems. The data showed that cell-free estimates of PDE activity seriously overestimated the intracellular rates of cAMP hydrolysis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Activation of adenylate cyclase in Acanthamoeba palestinensis.

Preincubation of Acanthamoeba palestinensis homogenates in 0.25M sucrose-TM (2mM MgSO4 and 5mM Tris-HCl, pH 7.4) at 0 degree C for increasing periods of time up to 3 h, leads to a progressive increase in the activity of adenylated cyclase. In contrast, preincubation of isolated membrane fractions enriched in enzyme activity in the same medium results in no activation. However, preincubation of membrane fractions in medium containing a high density of sugars (sucrose, glucose or fructose) mimics the activation obtained with homogenates. The high density sugar activation is time and temperature dependent, and reversible upon return to a low density medium. The high osmotic pressure of the sugars utilized may be a factor, since high concentrations of the sucrose polymer, Ficoll, which has low osmotic activity, causes not activation. Soluble activators, protein synthesis and changes in cyclic nucleotide phosphodiesterase activity were all eliminated as possible effectors of the apparent activation of adenylate cyclase. In contrast to mammalian adenylate cyclase, the endoplasmic reticulum localized enzyme of Acanthamoeba is inhibited by NaF and is unaffected by GTP, adenosine, epinephrine, prostaglandin E1, propranolol, and meclofenamic acid. These data indicate that the adenylate cyclase of Acanthamoeba is structurally different from that of most mammalian cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

A1 and A2 adenosine receptors regulate adenylate cyclase in cultured human lung fibroblasts.

Adenosine stimulates and inhibits adenylate cyclase activity and cAMP levels in WI-38 and VA13 fibroblasts. The inhibitory effects appear to be mediated by both A1 receptors and the P-site. Results supporting these conclusions are as follows: Adenosine by itself increased cAMP accumulation in these cells. PGE1-stimulated cAMP accumulation was inhibited by adenosine in a concentration-dependent fashion. IAP treatment blocked adenosine inhibition of cAMP accumulation and adenylate cyclase activity and enhanced adenosine stimulation of cAMP accumulation in VA13 cells. Theophylline and MIX attenuated adenosine inhibition of cAMP accumulation. Adenosine analogs with substitutions in the purine ring inhibited PGE1-stimulated cAMP accumulation and adenylate cyclase activity. PGE1-stimulated cAMP accumulation was inhibited by the P-site agonist 2'5'-dideoxyadenosine, but this inhibition was not attenuated by MIX or IAP treatment. These data support the idea that adenosine may inhibit cAMP accumulation in VA13 or WI-38 cells by acting at an A1 receptor of the P-site. The decrease in cAMP accumulation mediated by the A1 receptor appeared to be due at least in part to an Ni-mediated inhibition of adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1986

cAMP metabolism in an S49 mouse lymphoma variant with diminished phosphodiesterase activity.

This report presents the results of detailed examinations of cAMP metabolism in B1r, an S49 lymphoma protein kinase variant with very low phosphodiesterase activity. Among the conclusions reached are: As expected from the low phosphodiesterase activity previously reported, the cAMP turnover rate was relatively slow (t1/2 was 18-23 min at 37 degrees C). Basal cAMP levels ranged from about 1% to 5% of cellular ATP (i.e., 20-100 microM or 100-500 pmol/mg protein). These were many times higher than in most S49 wild type cells. The high basal cAMP levels made measurements of turnover in the absence of a hormone possible. The turnover constant for cAMP in unstimulated cells was 0.030 +/- 0.003 min-1. This was not significantly different than the value measured during epinephrine stimulation, which was 0.035 +/- 0.004 min-1. These turnover values were used to determine precise levels of adenylate cyclase activity throughout the time course of epinephrine stimulation. Desensitization was both rapid and profound, with the level of adenylate cyclase activity falling by 70% within the first 4 minutes of stimulation. This suggested that desensitization may be a very major factor in the attenuation of catecholamine action, at least in some cell types.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Phosphatidate and monooleylphosphatidate inhibition of fibroblast adenylate cyclase is mediated by the inhibitory coupling protein, Ni.

It has previously been shown that monooleylphosphatidate (MOPA) and phosphatidate inhibit cAMP accumulation in VA13 and WI-38 fibroblasts. In this study we investigated whether this inhibition might be due to a decrease in adenylate cyclase activity. Our results showed that both MOPA and phosphatidate inhibit prostaglandin E1-stimulated adenylate cyclase in WI-38 membranes in a concentration-dependent manner with half-maximal inhibitions at 0.1 and 0.5 microM, respectively, and maximal inhibitions of 35-55%. A 5 microM concentration of structurally similar lipids caused no significant inhibition. The inhibitory effects of MOPA and phosphatidate on adenylate cyclase were GTP dependent, greater at low concentrations of Mg2+, eliminated following treatment of cells with islet-activating protein, nonadditive with carbachol, and noncompetitive with prostaglandin E1. Collectively these data suggested that MOPA and phosphatidate inhibitions of cAMP accumulation were due at least in part to an Ni-mediated inhibition of adenylate cyclase. Furthermore, the inhibitions showed the same characteristics normally associated with hormonal inhibition of this enzyme.

Adenosine Diphosphate Ribose↗
PubMed1979

Adenosine 3',5'-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts.

Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10-30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 micronM prostaglandin E1 or 1 micronM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.

Cell Line↗
PubMed1979

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Effects of delta 1-tetrahydrocannabinol on cyclic AMP in cultured human diploid fibroblasts.

(-)-trans-delta 1-Tetrahydrocannabinol (delta 1-THC) antagonized the cyclic AMP responses of WI-38 fibroblasts to both prostaglandin E1 (PGE1) and catecholamines. Both cellular cyclic AMP accumulation and cyclic AMP escape to the incubation medium were reduced, but the reduction of escape was much more dramatic at all concentrations of the drug. Conversely, long term incubations of cells with delta 1-THC alone resulted in substantial accumulations of cyclic AMP in the incubation medium. This effect was potentiated by the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine and appeared to result from weak agonist activity of the cannabinoid as determined by a) stimulation of radioactivity incorporated into cyclic AMP using 3H-adenine prelabelled cells, and b) a rapid and pronounced increase in the activity ratio of cellular protein kinase. The antagonistic effect of delta 1-THC on the cellular response to PGE1 was greater in preconfluent cells than in confluent monolayers. Further, the increased sensitivity of preconfluent cultures to delta 1-THC was associated with the appearance of cytoplasmic vacuoles in the perinuclear region of the cells. Cannabidiol acted similar to delta 1-Thc in affecting cyclic AMP metabolis whereas cannabinol and cannabicyclol showed mixed effects on the various parameters studied.

1-Methyl-3-isobutylxanthine↗