Growth of S49 wild type cells in 3 nM epinephrine increases cyclic AMP phosphodiesterase activity.

In this communication we report that intact cell measurements of cAMP decay have shown an increase averaging over 80% in the cAMP decay constant (kdy) in intact S49 WT cells following a 24 hour growth in 3 nM epinephrine (24 hr/3 nM epinephrine). A comparable percentage increase was seen in cell free PDE activities from cells similarly treated. The enhanced PDE activity and kdy were detectable after 6 hours of incubation with 3 nM epinephrine, and were maximal by 24 hours of incubation. Lysate PDE activity returned to control levels within 24 hours after the cells were transferred to epinephrine-free growth medium. The increased PDE activity present in S49 WT cells after 24 hr/3 nM epinephrine was the result of increased concentrations of the enzymes already present or the expression of very similar enzymes, rather than the expression of enzymes with markedly different characteristics. Firstly, the apparent Km values for the PDE's in lysates from control and 24 hr/3 nM epinephrine cells were both in the region of 1 microM. Secondly, PDE activities in lysates from 24 hr/control and 24 hr/3 nM epinephrine S49 WT cells showed similar sensitivities to PDE inhibitors. There was support for the hypothesis that the increase in PDE activity in 24 hr/3 nM epinephrine S49 WT cells is dependent on the presence of cAPK. That is, although 24 hr/3nM epinephrine caused a distinct homologous desensitization of adenylate cyclase in S49 kin cells, neither kdy nor lysate PDE activity were affected. Additionally, 24 hours incubation of S49 WT cells with 3 microM dibutyryl cAMP resulted in increased PDE activity. Finally, the stimulatory effect of 24 hr/3 nM epinephrine on PDE activity was inhibited in the presence of cycloheximide, suggesting the involvement of protein synthesis in the process. This study shows that prolonged treatment with very low concentrations of epinephrine results in an increase in PDE activity which had a significant effect on cAMP accumulation. The increases were quantified by intact cell and cell free measurements, with kinetic data which were consistent with the hypothesis that the increased PDE activity in 24 hr/3 nM epinephrine cells reflected an increase PDE synthesis which was dependent on activation of the cAPK.

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.

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.

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.

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.

Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells.

We report here that, contrary to previously reported findings, treatment of S49 wild-type (WT) lymphoma cells with 0-50 nM epinephrine resulted in a heterologous desensitization of adenylyl cyclase (EC 4.6.1.1)--that is, epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase were reduced. Observation of this heterologous desensitization required the assay of adenylyl cyclase with submillimolar concentrations of Mg2+ and low concentrations of epinephrine. Also, whereas previously there had been no evidence for any role of cAMP-dependent protein kinase in the desensitization of the WT beta-adrenergic receptor, our data comparing the characteristics of the desensitization in WT, kin-, and cyc- lymphoma cells [where kin- and cyc- refer to variants of S49 WT cells lacking cAMP-dependent protein kinase activity (kin-) and the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (cyc-)] now suggest that cAMP-dependent protein kinase mediates the heterologous desensitization of adenylyl cyclase. Specifically, we found that only the WT cells exhibited epinephrine-induced heterologous desensitization. The kin- and cyc- cells exhibited only homologous desensitization, and much higher concentrations of epinephrine were required to elicit the homologous desensitization in the variants relative to the heterologous desensitization of the WT. Treatment of WT and cyc- cells with dibutyryl cAMP or treatment of WT with forskolin or PGE1 caused the heterologous desensitization of adenylyl cyclase, indicating that neither receptor occupancy nor activation of adenylyl cyclase was necessary for the heterologous desensitization.

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.

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.

Homologous desensitization of beta-adrenergic receptors in lymphoma cells is not altered by the inactivation of Ni (Gi), the inhibitory guanine nucleotide regulatory protein.

We had previously demonstrated that the cyc- mutant of S49 wild-type lymphoma cells both desensitizes and undergoes a sequestration-internalization of the beta-receptor in response to short-term treatment with adrenaline. The cyc- mutant of S49 wild-type lymphoma cells lacks the alpha s subunit of the stimulatory coupling protein Ns, but has fully functional Ni, the inhibitory component of the regulatory complex. This suggested that functional Ns was not required for desensitization. To examine the role of Ni in desensitization, both S49 wild-type and cyc- cells were treated with islet-activating protein under conditions that led to over 85% attenuation of Ni function in S49 wild-type cells and approx. 50% attenuation of Ni function in cyc- cells. This treatment had no effect on the adrenaline-induced desensitization of adenylate cyclase or the sequestration event measured by the apparent movement of beta-adrenergic receptors to a light-vesicle fraction. Further, the desensitization event, which occurs before the sequestration event, observable only in intact cells, was also not altered by islet-activating-protein pretreatment of S49 wild-type cells. The data suggest that a functional Ni is not required for desensitization in the S49 lymphoma cells.

Phorbol ester-induced augmentation and inhibition of epinephrine-stimulated adenylate cyclase in S49 lymphoma cells.

The effects of 4-beta phorbol 12-myristate 13-acetate (PMA) on hormone and forskolin-stimulated adenylate cyclase were evaluated in S49 lymphoma cells. Treatment of wild type (WT) S49 cells with PMA caused stimulation, inhibition or had no effect on epinephrine stimulation of cAMP accumulation. The effect observed was dependent on the length of PMA treatment, the concentration of PMA and the concentration of hormone (or forskolin) used to stimulate cAMP accumulation. Longer treatment times with PMA and higher PMA concentrations favored the inhibitory effects. Pretreating WT with 0.5 microM PMA for 18 min caused an increase in the EC50 and maximal levels for epinephrine stimulation of cAMP accumulation. Thus inhibition was seen at relatively low epinephrine concentrations and augmentation with high concentrations. The inhibitory effects of PMA on epinephrine-stimulated adenylate cyclase activity were observed only at low free Mg++ concentrations (0.75 mM). The effects of PMA on PGE1-stimulated cAMP accumulation were similar to those observed for epinephrine. In S49 WT cells 100 nM PMA augmented 5 microM forskolin-stimulated cAMP accumulation; however with 100 microM forskolin, PMA effects were minimal. PMA also attenuated Gi-mediated Gpp(NH)p inhibition of forskolin-stimulated adenylate cyclase in both WT and cyc- membranes, resembling the effects of pertussis toxin. The effects of various phorbol analogues on epinephrine-stimulated cAMP accumulation were as follows: 4 beta-phorbol 12,13-didecanoate had similar effects to PMA, 4 alpha-phorbol 12,13-didecanoate had no effects and 1-oleoyl, 2-acetylglycerol augmented epinephrine-stimulated cAMP accumulation at concentrations greater than or equal to 5 microM. Our results are consistent with a dual mechanism of PMA action on adenylate cyclase involving protein kinase C-mediated phosphorylation of Gi and of the beta-adrenergic receptor, the former leading to augmentation and the latter to inhibition of hormone-stimulated adenylate cyclase.

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.

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.

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.

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.

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.

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.

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.

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.