Desensitization and the control of cellular cAMP levels.
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Animals↗
Biomedical subjects
R Barber
Publications and source records attributed to R Barber.
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↗
Discrimination between intact cell desensitization and agonist affinity changes.
The relationship between epinephrine-induced receptor-epinephrine affinity changes and intact cell adenylate cyclase activity was investigated using S49 lymphoma cells. It was demonstrated that 20 nM epinephrine caused desensitization (defined as a reduction in the rate of cAMP synthesis with time), but no significant change in receptor-epinephrine affinity as measured by competition with [125I]iodopindolol. On the other hand, treatment with 5 microM epinephrine caused no desensitization (as defined above), but did cause a very significant reduction in receptor-epinephrine affinity. It is suggested that there is a desensitization process which is distinct from the agonist affinity shift and which is expressed primarily as a change in the EC50 of the hormone-induced activation. The demonstration of extensive desensitization at low concentrations of hormone may have important physiological implications.
Adenylyl Cyclases↗
Ethical issues in transplantation.
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Animals↗
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↗
Does left ventricular aneurysmectomy improve ventricular function in patients undergoing coronary bypass surgery?
Fourteen consecutive patients undergoing left ventricular aneurysmectomy and coronary artery bypass grafting were studied by multiple gated ventricular scintigraphy at rest and during exercise before and at six weeks and six months after surgery. All had congestive heart failure and 12 angina pectoris. Before operation left ventricular ejection fraction fell significantly with exercise, as did the regional wall motion score. Six weeks after surgery all surviving patients were free of angina, with an improvement in functional class; the total exercise workload improved significantly, but resting left ventricular ejection fraction was unchanged; the regional wall motion score improved in both the anterior and left anterior oblique projections, although extensive areas of abnormal contraction persisted. Exercise left ventricular ejection fraction improved significantly after operation at six weeks, and previous exercise induced abnormalities of regional contraction were abolished. Six months after operation angina pectoris had recurred in one patient, but there was no further change in ventricular function in the remainder. Although resting ejection fraction is not improved, symptoms, exercise workload, and exercise ventricular function can be improved by aneurysmectomy and coronary artery bypass grafting, but the respective contribution of these two procedures remains uncertain.
Adult↗
Adenylate cyclase activity as a function of forskolin concentration.
The activation of adenylate cyclase by forskolin was investigated in terms of the dissociation model of guanyl nucleotide binding protein (Ns). It was demonstrated that the biphasic forskolin concentration-response of adenylate cyclase could be explained by the dissociation of the beta subunit. The equations developed from such a theoretical approach gave an accurate description of concentration response data from S49 cultured mouse lymphoma cells.
Adenylyl Cyclases↗
Hormone and methylxanthine action on breast epithelial cells.
Human mammary carcinoma cells and normal mouse breast epithelial cells desensitized as the result of treatment with beta-adrenergic agonists. Accumulation of cAMP in the same cells was affected only slightly by caffeine and there was no detectable desensitization or hypersensitization as a result of that treatment. However, as in many other cell types, caffeine was an effective inhibitor of adenosine action. These observations do not support the hypothesis made by Minton and his co-workers (1), that treatment of breast epithelial cells with agents that increase cAMP accumulation leads to hypersensitization rather than desensitization.
2-Chloroadenosine↗
Effects of desensitization on the responses of WI-38 and S49 cells to hormones.
Experiments with intact cells were used to quantitate the effects of hormone desensitization on the cAMP accumulation in cultured fibroblasts (WI-38) and lymphoma cells (S49). Desensitization of WI-38 cells to prostaglandin E1 and epinephrine and of S49 cells to epinephrine was associated with a shift in the ED50 in each case to higher hormone concentrations. This led to the situation where cells under continuous stimulation by low concentrations of hormone demonstrated a greater relative decline in cAMP synthesis with time than the same cells treated with high hormone concentrations. Therefore, any general attempt to quantitate desensitization in terms of cAMP accumulation in these cells must take the hormone concentration of the assay into account.
Alprostadil↗
Desensitization and cyclic AMP turnover in S49 cells.
Measurements of cAMP accumulation and turnover have been used to quantitate desensitization of S49 wild type and variant cells to epinephrine stimulation. The extent of desensitization varied with the cell type and increased with temperature. Two traditional methods for detecting desensitization (the existence of a peak in the time course of accumulation or a diminution of the response to an agonist after prior exposure to the agent) did not always demonstrate its existence. That is, those methods always underestimated the degree of desensitization, sometimes to the extent of obscuring the phenomenon altogether.
Adenylyl Cyclases↗
beta-Adrenergic receptors and cyclic AMP responses to epinephrine in cultured human fibroblasts at various population densities.
The beta-adrenergic receptors of the intact human lung diploid fibroblast line Wl-38 and an SV-40 transformed clone of Wl-38, Wl-38-VA-13-2RA (VA13), were estimated in experiments utilizing the beta-adrenergic ligand, 125l-hydroxybenzylpindolol (125IHYP). When specific 125IHYP binding was measured in cells grown to relatively low population densities (0.15x10(6)cells/35mm dish), both Wl-38 and VA13 cells had approximately 40,000 beta-adrenergic receptors per cell. Wl-38 cells, when cultured to a high population density (0.5x10(6) cells/35/mm dish) had clearly diminished numbers of beta-adrenergic receptors and greatly decreased cAMP responses to epinephrine stimulation. On the other hand, in VA13 cells, neither the receptor number nor the beta-adrenergic response was affected by cell population density. In Wl-38 cells, the diminished cAMP response to epinephrine paralleled the decrease in number of beta-adrenergic receptors. Prostaglandin E1 (PGE1) stimulation of cAMP levels was unaffected by cell population density in either Wl-38 or VA13 cells. Thus, increased cell population density, perhaps related to density dependent inhibition of growth, caused a specific diminution in 125IHYP binding concomitant with decreased cAMP responses to epinephrine.
Cell Count↗
Differences in the forskolin activation of adenylate cyclases in wild-type and variant lymphoma cells.
The ability of the diterpene forskolin to stimulate cyclic AMP accumulation in intact cell and membrane preparations of wild-type S49 lymphoma cells (WT) and a number of variants has been confirmed. Additionally, a number of salient new findings have emerged: (a) A time delay in forskolin stimulation of cyclic AMP accumulation and adenylate cyclase (t 1/2 approximately equal to 1.5 min) occurred in all hormone-sensitive WT and variant cell and membrane preparations tested. (b) The time delay was missing in the adenylate cyclase-deficient variant (cyc-) of the S49 lymphoma cell, which also lacks functional adenylate cyclase-coupling proteins. (c) The simultaneous addition of epinephrine and forskolin to WT cells or to membrane preparations eliminated the time delay. (d) Forskolin stimulation of intact WT cells did not appear to desensitize adenylate cyclase. (e) The activation of WT adenylate cyclase by forskolin was biphasic with respect to concentration, with both high- and low-affinity components being apparent. In cyc-, only the low-affinity component was detected.
Adenylyl Cyclases↗
The effects of propranolol and acebutolol on the overnight plasma levels of anterior pituitary and related hormones.
1 The effects of single evening doses of the beta-adrenoceptor blocking agents propranolol (80 mg orally) and acebutolol (200 mg orally) on plasma levels throughout the night of prolactin, growth hormone, luteinising hormone, follicle stimulating hormone, cortisol and testosterone have been studied in seven healthy male volunteers. 2 Three way analysis of variance showed that acebutolol significantly reduced circulating levels of prolactin and follicle stimulating hormone, but did not alter the levels of the other hormones studied. 3 Propranolol significantly reduced follicle stimulating hormone and testosterone, and significantly increased circulating levels of cortisol, but caused no change in the other hormones studied. 4 Prolactin, luteinising hormone, testosterone and cortisol showed a significant variation with time indicating the existence of a diurnal rhythm in the pattern of their secretion. 5 There was a significant inter-subject variability in all the hormones studied. 6 There was a significant between-subject variation in response to both propranolol and acebutolol. 7 Different subjects showed significant variations with respect to time in prolactin, growth hormone and cortisol levels. 8 Neither propranolol nor acebutolol significantly altered the time course of secretion of any of the hormones studied. 9 Possible relationships of these beta-adrenoceptor blocker-induced changes in anterior pituitary and related hormones to the antihypertensive mechanism of acebutolol and propranolol are discussed.
Acebutolol↗
The quantitative relationship between intracellular concentration and egress of cyclic AMP from cultured cells.
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Adenine↗
Hypothesis: cyclic AMP turnover in S49 cells.
The fractional turnover constant (ke) of cAMP in S49 lymphoma cells stimulated by epinephrine was essentially identical to the decay constant for cAMP in these cells. This was in sharp distinction to the situation in the human diploid lung fibroblast WI-38, in which ke was much lower in hormone stimulated cells. In this study we report a new method for the determination of cAMP turnover. The method was based on the assumption that for tritium label to be incorporated into cAMP on treatment of cells with [3H]-adenine, the label must first pass through ATP. An equation relating the rate of change in cAMP specific radioactivity with cAMP and ATP specific radioactivities was thereby determined. The equation was expressed in a form that gave a linear graphical plot with the fractional turnover constant as the slope of the line.
Animals↗
Turnover of adenosine 3',5'-monophosphate in WI-38 cultured fibroblasts.
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Adenosine Triphosphate↗
Consequences of hormone-induced desensitization of adenylate cyclase in intact cells.
A hypothesis on the role of the hormone-induced desensitization of adenylate cyclase is proposed. It is suggested that the desensitization process could provide the cell with a highly efficient cyclic AMP system for transmitting hormone stimulus without requiring a large energy consumption. Theoretical considerations show that in fact the desensitization phenomenon allows the cyclic AMP system to present a good compromise between the efficiency and economy requirements of the cells.
Adenylyl Cyclases↗
The turnover of cyclic AMP in cultured fibroblasts.
The determination of the turnover of cAMP in WI-38 and VA13 cultured fibroblasts stimulated by prostaglandin E1 is reported. The method made use of data obtained from a process of continuously labeling the cellular adenine nucleotide pools by incubation with [3H]-adenine. The turnover of the cAMP was estimated from the delay in appearance of tritium label in the cAMP pool was compared to the cellular ATP. For WI-38 cells the half-life of cAMP when accumulation had reached a steady-state was 1.46 minutes; in the presence of 0.5 mM 1-methyl-3-isobutylxanthine (IBMX) the half-life was increased to 9.24 minutes. For VA13 transformed fibroblasts the half-life of cAMP determined by this method was 6.30 minutes. cAMP in these latter cells in the absence of hormone had a half-life of 3.01 minutes. This decrease supports the contention that the hormone has profound effects on phosphodiesterase as well as adenylate cyclase activities in these cells.
1-Methyl-3-isobutylxanthine↗