(6,9) were generously donated to our laboratory by Dr. Arthur J. Blume. ... 75-cm2 Corning tissue culture flasks containing Ham's F-10 medium. (Grand Island ...
THEJOURNAL OF BIOLOGICAL CHEMlSTRV Vol. 256, No. 13, Issue of July 10, pp. 6573-6576. 1981 Prrnted i n U.S A.
Cycloheximide Potentiationof Prostaglandin El- and Cholera Toxinstimulated Cyclic AMP Accumulation inNGlOS-CC15 NeuroblastomaGlioma Hybrid Cells* (Received for publication, January 16, 1981, and in revised form, March 9, 1981)
Robert D. Moylan and Gary Brooker From the Department of Pharmacology, University of Virginia School of Medicine, Charlottesuille, Virginia 22908
Previous studies have demonstrated that catecholamine responsiveness in a varietyof cells can be altered by inhibitors of RNA and protein synthesis. The neuroblastoma-glioma hybrid, NG108-CC15, which lacks of cyclic AMP, catecholamine-stimulated accumulation was investigated to determine if the responsiveness to prostaglandin El (PGEl) could be modified by inhibitors of protein synthesis. Cycloheximide in a time-dependent manner potentiatedthe ability of prostaglandin E1 to stimulate accumulation of intracellular cyclicAMP. However, the a-adrenergic inhibition of the prostaglandin response was not affectedby cycloheximide. Withdrawal of norepinephrine following a long-term incubation resulted in a potentiation of subsequent PGEIstimulatedcyclic AMP accumulation.Cycloheximide enhanced this norepinephrine withdrawal effect. Our previous studies have shown that choleratoxin induces refractoriness to p-adrenergic agonists in C62B rat astrocytoma cells andthatcycloheximide blocked this action of cholera toxin. In an analogous manner cholera toxin caused refractoriness to subsequent prostaglandin-stimulated cyclic AMP production in NGlOS-CC15 cells, and cycloheximide reduced cholera toxin-induced prostaglandin refractoriness. Thus cycloheximide potentiates the prostaglandin stimulatory effect, has no effect on the abilityof a-agonists to inhibit the prostaglandin response, increases the stimof PGEl afternorepinephrine withulatoryeffect drawal,andreducescholeratoxin-induced PGEl refractoriness.Theseobservations suggest that PGE1stimulated cyclic AMP accumulationin NGlO8-CC15 cells contains components which are regulated by de nouo protein synthesis.
accumulation. The NG108-CC15hybrid is an example of such a cell. Prostaglandin El stimulation results in the accumulation of cyclic AMP. When PGE1’ and norepinephrine are added concurrently, however, norepinephrine inhibits PGEIstimulated accumulation of cyclic AMP (6, 7). If NG108-CC15 cells are pretreated with norepinephrine for a few hours and the norepinephrine is removed, both basal and PGEI-stimulated cyclase activities are markedly increased (8). Thus it appears that PGElstimulation of NG108-CC15 cells is subject to both inhibition and potentiation by a-adrenergic receptors. The experiments presented here show that NG108-CC15 cell responsiveness to PGElstimulation is also regulated by mechanisms which workindependently of hormone receptor interactions. EXPERIMENTALPROCEDURES
Cell Culture-NG108-CC15 neuroblastoma-glioma hybrid cells derived from a clone as described by Traber et al. (6,9) were generously donated to our laboratory by Dr. Arthur J. Blume. The cells were maintained at 37 “C in a humidified atmosphere of 5% C02-95%air in 75-cm2Corning tissue culture flasks containing Ham’s F-10 medium (Grand Island Biological Co.) with 10%fetal calf serum (Grand Island M hypoxanthine, 4 X 10” M aminopterin, and Biological Co.) and 1.6 X M thymidine. For the experiments the NG108-CC15 cells were trypsinized (0.25% trypsin, Grand Island Biological Co.) in saline solution (NaC1, 137 mM, KCI, 5.4 mM, KH2P04, 1 mM, Na2HP04, 1 mM) and replated in 1 ml of Ham’s F-10 medium containing 10% donor calf serum (Flow Laboratories) on 16-mm cluster culture dishes (Linbro) at a density of 3 X lo5cells/ml. On the day preceding the experiment, the culture dishes were changed to fresh Ham’s F-10 with 10% donor calf serum. Immediately before drug additions the cells were changed to Ham’s F-10 medium without serum. All incubations were done at 37 “C in a humidified atmosphere. Cyclic AMP Assay-The cyclic AMP content of NG108-CC15 cells was measured following drug treatment by rapidly aspirating o f f the culture containing medium and treatingthe cells adhering to theplate with 0.1 M HCl. The culture dishes were then agitated on an orbital The regulation of hormone-stimulated cyclic AMP accu- shaker for 20 min a t room temperature. The HCl extract of the cells mulation in cells has been the subject of intensive investiga- was removed from the plates with Pasteur pipettes and stored frozen assayed. Cyclic AMP was assayed using the Gamma-Flo autotion. Receptor-mediated increases incyclic AMP are self- until mated radioimmunoassay system (10,11) using the acetylation techlimiting in a variety of tissues. The loss of responsiveness to nique of Harper and Brooker (12). repeated rechallenge with an agonist commonly referred to as Protein content of the cluster wells was determined in 0.2 M NaOH refractoriness occurs with a loss of receptors (1-3) or in some extracts by an automated Lowry (13) procedure using bovine serum cases occurs through events which appear to require cyclic albumin as the standard. Materials-Prostaglandin E,, norepinephrine, cycloheximide, and AMP, are independent of receptor loss, and are reversed or prevented by protein synthesis inhibitors (4,5). In addition to actinomycin D were obtained from Sigma. Phentolamine (regitine mesylate) was a generous gift from Ciba Pharmaceuticals. I-Methylthe self-limiting effects of refractoriness some ceus possess 3-isobutylxanthine was obtained from Calbiochem. The cholera enreceptors which whenstimulated with the appropriate agonist terotoxin was obtained from Schwarz/Mann.
inhibit the ability of other agonists to stimulate cyclic AMP
RESULTS
* This work wassupported by National Institutes of Health Grants Cycloheximide Potentiation of PGE, Response in NG108HL15985, AM22125, and HL19242. The costs of publication of this CC15 Cells-Preincubation ofNG108-CC15 cells with the article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord’ The abbreviations used are: PGEl, prostaglandin El; MIX, 1ance with 18 U.S.C. Section 1734 solely to indicate this fact. methyl-3-isobutylxanthine.
6573
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Cycloheximide Potentiation
protein synthesis inhibitor cycloheximide at a concentration 1 of 5 p g / d for 5 h results in a 4-fold increase in the accumulation of cyclic AMP at maximal doses of PGEl (Fig. 1).In the presence of the cyclic nucleotide phosphodiesterase inhibitor, methyl isobutylxanthine,the maximal response to PGEl is increased 2-fold when compared to cells treated only with PGEl. Incells pretreated with 5 pg/ml of cycloheximide for 5 h a challenge with PGEl andMIX causes a doubling of cyclic AMP accumulation over that found in cells stimulated by PGE, alone. Cycloheximide did not significantly change the apparent K , for PGEI. The possibility that cycloheximide decreased the synthesis of a rapidly turning over phosphodiesterase was ruled out by the results of direct phosphodiesterase measurements in 5 homogenates each from control cells and cells treated with 5 pg/ml of cycloheximide for 6 h at 37 "C. Phosphodiesterase was measured in 25 mM Tris-HC1, 5 mM MgCl?, 1mM EDTA, and 1 ~ L Mcyclic AMP. The rate of cyclic AMP disappearance was automatically determined at 5-min intervals over 40 min by the Gamma-Flo automated radioimmunoassay for cyclic AMP (10, 11).A linear decline uersus time (correlation coefficients ~ 0 . 9 9in ) cyclic AMP concentration was observed with no difference between extracts from control (309 k 5 pmol/ mg/min) and cycloheximide (294 +- 6 pmol/mg/min) treated 0' I cells. 1 2 3 4 5 6 Time Dependence of Cycloheximide Potentiation of PGE, HOURS Response in NGZO8-CCZ5 Cells-The addition of cycloheximide (5 pg/ml) increasesthe PGE1-mediated accumulation of FIG. 2. Time course of the effect of cycloheximide and acticyclic AMP in NG108-CC15cells in a time-dependent manner.nomycin D on the potentiation of PGE, response in NG108Following treatment with cycloheximide for 6 h the accumu- CC15 cells. NG108 cells were pretreated forthe times indicated with lation of cyclic AMP in these cells is increased about &fold either cycloheximide ( 5 pg/ml), actinomycin D ( 5pg/ml), or incubated over that of PGE, without cycloheximide (Fig. 2). In control without drugs in Ham's F-10 medium (control). The cells were challenged for 20 min with 1 (LMPGEI.Data represent mean k S.E. for 4 cells there was a 28% decline in PGEI-stimulated cyclic AMP determinations per condition. accumulation after 6 h and a 9% decline at 4 h. The PGEl response in cells treated with cycloheximide for 4 h or 6 h was increased about 2-fold and %fold, respectively, when compared with PGEl stimulation in cells treated with cycloheximide for 50 min. Actinomycin D at 5 pg/ml increased the PGEl-stimulated cyclic AMP response about 50% in NG108CC15 cells at 50 min, the fwst time point measured. This increased PGEl response in the presence of actinomycin D remained constant over 6 h and is presumably independent of its ability to inhibit RNA synthesis. Modification of PGEl-stimulatedCyclic AMP Accumulation in NG108-CC15 Cells by Pretreatment with Norepinephrine: The Effects of Cycloheximide-Sabol and Nirenberg (8) previously have shown that treatment of NG108-CC15 cells with norepinephrine followed by abrupt withdrawal increases PGE1-stimulatedadenylate cyclase activity in membrane preparations from these cells. The data in Fig. 3 show that pretreatment of NG108-CC15 cells with norepinephrine for 2 or 4 h followed by withdrawal and challenge for 20 min with 1 p~ PGEl causes a 2-fold increase in cyclic AMP in the cells compared to the PGE, response seen in untreated control cells. Cycloheximide pretreatment of the cells for 2 h increases the PGElresponse in NGlO8-CC15 cells to 150% ofthe control level. When NG108-CC15 cells were pretreated simultaneously with norepinephrine and cycloheximide and norepinephrine subsequently withdrawn, a 300%increase at 2 h and FIG. 1. Potentiation of PGEI-mediated cyclic AMP accumulation in NGlO8-CC15 cells. Cells were first incubated for 5.25 h a t 665% increase at 4 h was observed in PGEI-mediated cyclic AMP accumulation. 37 "C and subsequently challenged for 20 min with varying concentrations of PGE, alone (control, M j or in the presence of 0.1 mM The Effect of Cycloheximide on Cholera Toxin-induced 1-methyl-3-isobutylxanthine (MIX, M j . Other cells were first Refractoriness to PGE, Stimulation inNG108-CC15 Cellsincubated for 5.25 h with 5 p g / d of cycloheximide and subsequently Preincubation with cholera toxin for 4 h causes the loss of challengedfor 20 min withvarying concentrations of PGEl (CH, PGE1-stimulated cyclic AMP accumulation in NG108-CC15 M), or in the presence of 0.1 mM 1-methyl-3-isobutylxanthine (CH + MZX, H). Eachpoint is the meanof 3-4 plates. Variance cells as shown in Table IA. The basal accumulation of cyclic AMP in cholera toxin-treated cells is doubled while the PGEI( 7 6 S.D.) was less than 10%.
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Cycloheximide Potentiation
cells pretreated with cholera toxin. After a 4-h pretreatment with both cycloheximide and cholera toxin, the basal response to cholera toxin is elevated by 113% over cells treated with cholera toxin alone, and the PGE, response is elevated by 202%. Norepinephrine Acutely Inhibits the Cycloheximide-mediated Increase in PGE, Responsiveness in NG108-CC15 Cells-The acute inhibitory effect of norepinephrine on PGEI responsiveness in NG108-CC15 cells is mediated by a-adrenergic receptors and has been described in detail (7). The experiments in Table IB test theeffects of norepinephrine on the responsiveness of cells that have been pretreated with cycloheximide, cholera toxin, and cycloheximide plus cholera toxin. As shown in Table 1B norepinephrine reduces the prostaglandin-stimulatedaccumulation ofcyclic AMP by about 90%compared to untreatedcontrols (Table IA).Cycloheximide greatly potentiates the PGEl-stimulated accumulation of cyclic AMP as seen inTable IA. This response is again reduced by90% (319 versus 2982 pmol/mg of cyclic AMP) following an acute treatmentwith norepinephrine. The inhibitory effect of norepinephrine is also apparently additive with the inhibitory effect of cholera toxin on the CH JE PGEl response in NG108-CC15 cells.Pretreatment with chol+ era toxin followed by a challenge with norepinephrine and :H PGEl abolishes the PGE,-mediated accumulation ofcyclic AMP in these cells. Norepinephrine also inhibits the cholera 2h 4h toxin-stimulated increase in basal cyclic AMP accumulation. FIG. 3. Potentiation of norepinephrine withdrawal effects The ability of cycloheximide to increase the response of on PGE1-stimulated cyclic AMP accumulationin NG108-CC15 cells by cycloheximide. NG108-CC15 cells were pretreated for 2 NG108-CCl5 cells to cholera toxin and to increase the PGEl and4h with Ham's F-10 medium (Con) or medium containing response in cells made refractory by cholera toxin is inhibited norepinephrine ( N E ) M), cycloheximide ( C H ) (5 pg/ml), or by norepinephrine.
t
+
E
I
--
norepinephrine and cycloheximide ( N E + CHI. At the end of the pretreatment the cells were washed with fresh medium, and cycloheximide was added to cells previously treated with cycloheximide. M phentolamine was added to plates which previously received norepinephrine. The figure shows the accumulation of intracellular cyclic AMP for each of the preincubation conditions following a 20min challenge with 1.0 PM PGE, as mean -C S.E. of 4 determinations per treatment.
TABLEI The effect of cycloheximide on PGE, response in NGIOS-CC15 cells following cholera toxin treatment and acute norepinephrine challenge NG108-CC15 cells were incubated for 4 h in Ham's F-10 medium (control) or Ham's F-10 containing cycloheximide (5 pg/ml), cholera toxin (3 nM), or cycloheximide plus cholera toxin. At the end of 4 h the cells were challenged with I p~ PGEl (Group A). In Group B all of the cells were treated with norepinephrine (10 ,LAM)during the final 20 min of incubation. Basal 1 FM PGEI" pnol/cAMP/mg protein
A Control 72 1- 3 Cholera toxin 168 1- 5 Cycloheximide 80 1- 4 Cholera toxin + cycloheximide 358 k 29 Bb Control 65 1- 4 Cholera toxin 80 f 4 74 f 4 Cycloheximide Cholera toxin + cycloheximide 118 & 10 20-min challenge. * In the presence of 10 PM norepinephrine.
*
774 49 221 f 6 2982 f 153 668 f 65 164f4 93 f 5 319 16 157 k 7
*
stimulated levels are inhibited by 70% compared to controls. The cholera toxin-mediated loss of PGEl stimulation is maximal by 4 h.2 Cycloheximide increases the responsiveness of NG108-CC15 R. D. Moglan and G. Brooker, data not shown
DISCUSSION
In this study theprotein synthesis inhibitor cycloheximide was found to potentiate the PGEl-mediatedaccumulation of cyclic AMP in NG108-CC15 hybrid cells. In the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine the level of cyclic AMP in the cells was increased over cells treated with PGEl alone. Cycloheximide pretreatment potentiated the response of cells treated simultaneously with both MIX and PGE,. Furthermore, direct measurement of phosphodiesterase activityin crude homogenatesfrom control and cycloheximide-treated cells showed no change in phosphodiesterase activity. These findings suggest an action of cycloheximide occurring at some site other than that involving the degradation of cyclic AMP. The potentiation of the PGEl stimulation of the NG108CC15 cells is dependent on a preincubation with cycloheximide. At time points less than 1 h cycloheximide was without effect on the responsiveness to PGEl.2Significant alterations in PGE,-stimulated accumulation of cyclic AMP are not seen until 1to 2 h of incubation with cycloheximide. These findings reinforce the view that PGEl responsiveness in NG108-CC15 cells may be increased by an inhibition of protein synthesis. The RNA synthesis inhibitor actinomycin D causes a significant increase in PGEl-mediated cyclic AMP accumulation even at early time points as shown in Fig. 2. In contrast to cycloheximide this effect does not increase with time. While it is possible that actinomycin D could exert its effect by preventing the de novo synthesis of protein, the lack of time dependence and the earlyplateau of its effect make this unlikely. From these studies it appears that the NG108-CC15 cells may be regulated by mechanisms which involve long term compensatory increases in hormonal responsiveness that are both dependent and independent of protein synthesis. We have foundthat cycloheximide potentiates the norepinephrine
6576
Cycloheximide Potentiation
withdrawal effect in whole cells challenged with PGE,. Sabol and Nirenberg (8) previously found that cycloheximide had no effect onthe development of the PGEI-stimulated responsiveness after norepinephrine withdrawal in their cyclase preparations from NGlOS-CC15 cells and used this finding to suggest that increased PGEl cyclase responsiveness occurred in the absence of de novo protein synthesis. Our findings in whole cells suggest an additional effect of cycloheximide on PGEI responsiveness that is additivewithnorepinephrine withdrawal. Recently Nickols and Brooker (16) have shown that protein synthesisinhibitors potentiatethe cholera toxin-mediated accumulation of cyclic AMP in cultured cells. Additionally they have found that cholera toxin induces refractoriness to isoproterenol stimulation in C6-2B cells and that this refractoriness is prevented by cycloheximide (17). We have found that the responsiveness of NG108-CC15 cells to PGE, stimulation is reduced when the cells are pretreated with cholera toxin. This observation is similar to that seen in C6-2B cells (17) and may suggest a mechanism of refractoriness that is initiated by the intracellular accumulation of cyclic AMP or an action of cholera toxin upon one of the components of the adenylate cyclase system (18). The partial reversal of this cholera toxin-induced refractoriness by cycloheximide suggests that theloss of responsiveness to PGElfollowing cholera toxin treatment is mediated inpart by events requiring protein synthesis. In the NG108-CC15 cells, acute inhibitory effects on the PGE1-stimulatedaccumulation of cyclic AMPhave been found for opiate (19), muscarinic (9, 20) and a-adrenergic agonists (7).Our studies show that thea-adrenergic inhibition of PGE1-stimulated cyclic AMP accumulationin NG108-CC15 cells is not reversed by prior treatment with the protein synthesis inhibitor cycloheximide. Moreover, the partial reversal by cycloheximide of cholera toxin-induced refractoriness to PGE, stimulation is inhibited by the a-adrenergic effects of norepinephrine. These findings suggest that the amediatedinhibition of PGEl stimulation andthe cholera toxin-induced refractoriness in NG108-CC15 cells are separable inhibitory effects. Previous studies from our laboratory have shown that the exposure of the rat astrocytoma cell line C6-2B to agents which elevate intracellular cyclic AMP induces a time-dependent refractoriness in these cells to further accumulation of cyclic AMP following a rechallenge with the &adrenergic agonist isoproterenol (4,5,16,17).Protein and RNA synthesis inhibitors are effective in reducing the degree of refractoriness developed in the C6-2B cells and have been shown to prevent or reverse refractoriness in other cell and tissue preparations (14-16). When C6-2B cells are incubated with proteinsynthesis inhibitors alone nochange is observed in hormonal responsiveness compared to controls (4). These datasuggest that the primary effect of protein synthesis inhibitorsis to prevent the development of refractoriness after an initial period of elevated cyclic AMP in C6-2B cells. Presumably the de nouo synthesis of a protein which interferes with the hormonal stimulation of C6-2B cells is blocked by this maneuver. In contrast toC6-2B cells NG108-CC15 cells show an enhanced
hormonal response following a pretreatment with cycloheximide alone. These findings imply that NG108-CC15 cells possess protein constituents which modify the cells’ responsiveness to agents that increase intracellularcyclic AMP. It is also possible that the synthesis of an inhibitory modifier is increased by prolonged exposure toagents which elevate intracellular cyclic AMP. This would explain why cholera toxin is capable of inducing refractoriness to PGElstimulation in NG108-CC15 cells. The partial reversal of cholera toxininduced refractoriness by cycloheximide in these ceils is evidence that protein synthesisis involved in the development of refractoriness. The fact thatprolonged exposure to cycloheximide increased PGE, responsiveness in control NG108-CC15 cells suggests that this modifier is being synthesized and degraded at some basal rate. Ultimately the difference in the ability of cell lines, e.g. C6-2B and NG108-CC15, to accumulate cyclic AMP in response to agonist stimulation may be, reflected by the basal rates of synthesis of regulatory protein(s). Acknowledgments-We greatly appreciate the expert and meticulous technical assistance of Carolyn Pedone and Anne O’Brien. Celinda Johnson skillfully assisted in the preparation of the manuscript.
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