Proc. Nat. Acad. Sci. USA Vol. 71, No. 1, pp. 3844-3848, October 1974 Cyclic AMP-Mediated nduction of the Cyclic AMP Phosphodiesterase of C-6 Glioma Cells (dibutyryl cyclic AMP/norepinephrine/norepinephrine refractoriness) JOAN P. SCHWARTZ AND JANT V. PASSONNAU Section on Cellular Neurochemistry, Laboratory of Neuropathology and Neuroanatomical Sciences, National nstitute of Neurological Diseases and Stroke, National nstitutes of Health. Bethesda, Maryland 214 Communicated by Carl F. Cori, July 11, 1974 ABSTRACT Long-term regulation of the cyclic nucleotide phosphodiesterase of the C-6 rat glioma cell line has been studied. Both the low Km and high Km activities can be induced by elevation of intracellular cyclic AMP levels following either dibutyryl cyclic AMP or norepinephrine treatment of the cells. The enzymes are maimally induced by 3-4 hr. The presence of either cycloheide or actinomycin D prevents induction by either dibutyryl cyclic AMP or norepinephrine. vidence is presented that the norepinephrine effect is mediated by the ft-catecholamine receptor. The increased phosphodiesterase activity causes a partial refractoriness to a second challenge with norepinephrine, which can be overcome by blockade of the induction with cycloheimide. The results suggest that just as short-term regulation of cyclic AMP levels occurs via changes in the rates of synthesis or degradation, longterm alterations of the system may also involve either the adenylate cyclase or the phosphodiesterase. ntracellular levels of cyclic adenosine-3': 5'-monophosphate (camp) can be regulated in several ways: by alterations in the rate of synthesis, of degradation, or of ecretion. The C-6 rat glioma cell line has been shown to respond to norepinephrine (N) or other fl-agonists with adenylate cyclase activation (1) and a resultant elevation of intracellular camp levels up to 25-fold (2). Both low Km (1.5,gM) and high Km (2 1M) cyclic AMP phosphodiesterase activities are present in the cells (1) and both activities are inducible by treatment of the cells with dibutyryl (Bt2)cAMP (1). A similar induction of phosphodiesterase by Bt2cAMP has been demonstrated in other cell lines (3-6). This suggests that camp levels may be subject not only to short-term regulation by hormone stimulation of the adenylate cyclase but also to long-term regulation by increased activity of the degradative enzyme phosphodiesterase. Recent evidence suggests that elevated phosphodiesterase activity may be at least partially responsible for the refractoriness to a second addition of N observed in the C-6 glioma cells. Thus the smaller rise in camp levels following a second application of N could be partially overcome by concomitant addition of a phosphodiesterase inhibitor, either papaverine or isobutylmethylanthine (7, 8). n this paper, we present evidence that either Bt2cAMP or N treatment of the C-6 glioma cells results in an induction of both low and high Km phosphodiesterases which is detectable within 3 min and maimal by 3-4 hr. The induction requires both protein and RNA synthesis. The N effect is prevented by compounds that block Abbreviations: N, norepinephrine; Bt2cAMP, N6,2'-dibutyryl adenosine-3': 5'-monophosphate. 3844 3-catecholamine receptors. The addition of cycloheimide with the first N treatment is shown to prevent not only the rise in phosphodiesterase activity but also the refractoriness to a second N treatment. A preliminary report of these results has been presented (9). MATRALS AND MTHODS Cell Culture. The C-6 glioma cell line was obtained from the American Type Culture Collection. The cells were grown in Falcon plastic flasks (25 cm2) with Ham's F-1 medium containing 1% fetal calf serum (Grand sland Biological Co.) in a humidified atmosphere of 95% air-5% CO2 at 37. All subcultures contained 33 units/ml of penicillin G and 5,g/ml of streptomycin. Other details were as previously described (1). Phosphodiesterase Assay. 3': 5'-Cyclic-AMP phosphodiesterase (C 3.1.4.17) activity was determined according to the method of Thompson and Appleman (1), using cells suspended by incubation with.5 mm ethylenediaminetetraacetate (DTA) in balanced salts solution and resuspended in 5 mm imidazole, ph 7.6, containing 3 mm MgCl2 and 25 ug/ml of alkaline phosphatase. The low Km activity was assayed at 1.4M&M camp and the high at 1 mm camp, using [3H]cAMP. [3H]Leucine ncorporation. Cells were incubated in fresh Ham's F-1 containing 1.25 1Ci (.42,Ci/ml) of [3H]leucine. At the end of the incubation, the cells were suspended as described above and pelleted at 1 X g, and the pellet was taken up in 1 ml of 5% trichloroacetic acid. The precipitate was washed three times with 2-ml portions of trichloroacetic acid and finally dissolved in 5 ul of 88% formic acid. One aliquot was assayed for protein by the method of Lowry et al. (11), while radioactivity was determined in another, by means of a Packard Tri-Carb liquid scintillation spectrometer. Cyclic AMP Assay. Cyclic AMP was assayed by a modification (12) of the Gilman binding assay (13). Materials. Materials were purchased as follows: Sigma Chemical Co.: alkaline phosphatase (type ), D-propranolol HCl, cycloheimide, iarterenol bitartrate, Bt2- - cyclic AMP (monosodium salt), actinomycin D; Boehringer Mannheim Corp.: cyclic AMP; New ngland Nuclear Corp.: [3H]cyclic AMP (24.1 Ci/mmole), [3H]leucine (5. Ci/- mmole); Aldrich Chemical Co.: dichloroisoproterenol * HCl. We thank Smith, Kline and French Co. for the donation of - phenoybenzamine HC1.
nduction of Cyclic AMP Phosphodiesterase 3845 co Q 1 Ds 1 - F am OD _W a- -J. w 4C- ~ -J a- : 2 4 HOURS WTH Bt2cAMP + THOPHYLLN FG. 1. nduction of cyclic AMP phosphodiesterase (PD) by dibutyryl-cyclic AMP plus theophylline. The cells were grown as described under Materials and Methods. On the day of the eperiment, the old medium was removed and the cells were washed two times with 3-ml aliquots of Hanks' balanced salts solution and incubated at 37 in 3 ml of Ham's F-1 containing 1 mm Bt~cAMP + 1 mm theophylline. At the indicated times, the cells were suspended and assayed for phosphodiesterase activity as described under Materials and Methods. ach point represents the mean of four dishes. The eperiment is one representative of three eperiments. RSULTS Previous eperiments had shown that growth of the C-6 glioma cells in the presence of 1 mm Bt2cAMP plus 1 mm theophylline for 4 hr resulted in elevated cyclic AMP phosphodiesterase activity (1). Fig. 1 illustrates that within 4 hr of incubation with 1 mm Bt2cAMP plus 1 mm theophylline, both the low and high Km activities have doubled. Sodium butyrate, at 1 mm, had no effect on enzyme activity (data not shown). The results shown in Table 1 indicate that although there is a partial increase in activity when the cells are treated with.3 mm Bt2cAMP in the presence of 1 mm theophylline, 1. mm has a greater effect. Larger doses of Bt2cAMP were not used because they adversely affected cell growth. Also shown in Table 1 are the effects of cycloheimide, a drug that blocks protein synthesis, and actinomycin D, a drug that blocks RNA synthesis. When either drug was included in the 4-hr incubation with Bt2cAMP, the effects of Bt2cAMP were completely inhibited. However, when cycloheimide or actinomycin D was added without Bt2cAMP, there was no change in enzyme activity. The dose of cycloheimide utilized was that dose which blocked overall protein synthesis appro. 95% (data not shown). These results demonstrate that the increase in phosphodiesterase activity produced by Bt2cAMP requires both new RNA and protein synthesis. Since norepinephrine can elevate the C-6 glioma cell camp content (2), we net asked whether a long-term effect of this rise in camp might be phosphodiesterase induction. ncubation of the cells with.1 mm N resulted in increasing phosphodiesterase activity with time (Fig. 2). By 4 hr, both the low and high Km activities are maimally elevated. Halfmaimal induction was achieved with 5-1 pm N (results not shown). The results are very similar to those obtained for 2 3 4 5 HOURS WTH NORPNPHRN FG. 2. nduction of cyclic AMP phosphodiesterase (PD) by norepinephrine. The eperiment was carried out as described in the legend for Fig. 1, ecept that the cells were incubated in 3 ml of Ham's F-1 containing.1 mm norepinephrine. ach point represents the mean of four dishes. The eperiment is one representative of four eperiments. Bt2cAMP treatment (Fig. 1), although N caused a faster rate of increase in activity. n addition, enzyme activity starts to fall off after 4-6 hr with N, whereas it remains elevated as long as the Bt2cAMP concentration is maintained. That this increase also requires new protein and RNA synthesis is shown in Table 2. Again, the presence of either cycloheimide or actinomycin D during the incubation with N prevented the rise in enzyme activity. Also shown in this table are cpm of [13Hleucine incorporated per jig of total cellular protein during the course of the incubation. While N has no effect on the relative incorporation, cycloheimide inhibited it appro. 92%. Actinomycin D had no effect on [3H11leucine incorporation. TABL 1. nduction of the phosphodiesterase by dibutyryl-cyclic AMP + theophylline and its blockade by cycloheimide or actinomycin D Cyclic AMP phosphodiesterase Treatment Low Km High Km (4-hr incubation) activity activity (pmoles/mg of protein per min) Control 32.9 i 3.1 369 + 4.1 mm Bt2cAMP + 1 mm theophylline 3.6 A 3.3 488 4+ 62.3 mm Bt~cAMP + 1 mm theophylline 51.3 + 2.8 67 +L 5 1. mm Bt2cAMP + 1 mm theophylline 75.1 + 5.3 78 + 72 1. mm Bt2cAMP + 1 mm theophylline + 25 ig/ml of cycloheimide 3.7 + 3.3 233 ± 23 1. mm Bt2cAMP + 1 mm theophylline + 5 pg/ml of actinomycin D 3.6 ± 1.2 18 + 24 The eperiment was carried out as described in the legend to Fig. 1. The results represent the mean + SM for n = 8.
3846 Biochemistry: Schwartz and Passonneau TABL 2. nduction of the phosphodiesterase by norepinephrine and its blockade by cycloheimide or actinomycin D camp phosphodiesterase Low Km High Km [3H]Leu activity activity incorporation Treatment (pmoles/mg of (cpm/pg of (3-hr incubation) protein per min) protein) Control 3.8 ±- 4.1 35 ± 1 7.11 ±-.58.1mMN 91.3 ±4.1 7± 3 6.95 ± 1.17.1 mm N + 25 jug/ml of cycloheimide 51.6 ±t 3.2 363 ± 27.59 ±.7.1 mm N + 5 pg/ml of actinomycin D 43. ± 6. 386 ±- 69 8.18 ±.74 The eperiment was carried out as described in the legend to Fig. 1. The [3H]leucine incorporation was done as described under Materials and Methods. The results represent the mean ± SM for n = 4. The eperiment is one representative of three eperiments. Since N can interact with both a and 13 receptors, we eamined which type was involved in this enzyme induction. ither dichloroisoproterenol or propranolol, both 13-receptor antagonists, was found to block the induction of -phosphodiesterase by N, whereas the a-blocker phenoybenzamine had almost no effect (Table 3). Both,1-blockers also prevent the rise in camp, while the a-blocker does not (data not shown). These results suggest that N is acting via the 13- receptor. This concept is further supported by the finding that isoproterenol, a pure 13-agonist, causes a similar enzyme induction (data not shown). The above results suggest that an elevation of intracellular camp levels in the C-6 cells, whether produced by incubation with Bt2cAMP or N, mediates the increase in cyclic AMP phosphodiesterase observed. Further evidence for this suggestion is shown in Fig. 3. n this eperiment, cells were incubated with N for zero to 5 min. Some cells were fied after this incubation in order to determine camp levels, while others were incubated an additional 3 hr without N, follow- TABL 3. ffect of a- and,-blockers on norepinephrine induction of the phosphodiesterase camp phosphodiesterase Low KmaHigh Km Treatment activity activity (3-hr incubation) (pmoles/mg of protein per min) Control 28.9 i.1 348 i 1 5 AM N 69.3 i 13.2 736 i 6 5 MM N + 1AM dichloroisoproterenol 46.3 i 2.7 449 i 37 5 MM N + 1 M propranolol 37. i 1.9 29 i 4 5 AM N + 1 AM phenoybenzamine 62.7 i 5.1 642 i 33 The eperiment was carried out as described in the legend to Fig. 1. The results represent the mean i SM for n = 4. The eperiment is one representative of three eperiments. ing which phosphodiesterase activity was measured. The results demonstrate that with increasing time of eposure to N, camp levels are increasingly elevated. The relative increase of low Km phosphodiesterase activity corresponded closely with the relative elevation of camp levels, although maimal induction of the enzyme apparently occurred with camp levels above 1 pmoles/mg of protein. Similar results were obtained for the high Km enzyme. Readdition of N to cells 4 hr after an initial incubation with the drug results in a much smaller elevation of camp levels (8), relative to the rise seen with the first addition of N. n order to determine whether this refractoriness might be partially eplained by the increased phosphodiesterase activities measurable at this time, we carried out the eperiment shown in Table 4. Cells were incubated for an initial 4-hr period followed by a second 1-min incubation. The presence of N during the first 4-hr incubation resulted in the epected rise of phosphodiesterase activity. This rise was blocked by the additional presence of cycloheimide during this first incubation. N addition during the second (1-min) incubation caused camp levels to rise to 1711 pmoles/mg of protein, versus 12.2 in control cells. f the N were present during only the first incubation, the camp level was still appro. 5-fold TABL 4. Correlation between phosphodiesterase activity and refractoriness of the cells to a second norepinephrine treatment Treatment camp phosphodiesterase 4-hr 1-min Low Km activity High Km activity cyclic AMP incubation incubation (pmoles/mg of protein per min) (pmoles/mg of protein) - 32.8 ±t 1.6 36± 3 12.2 ±: 1. N 1711 ± 23 N - 78.1 ± 6.6 74 ± 49 57.2 ± 7.1 N N 265 ± 12 N + CH - 42. ±t 1.7 416 ± 49 57.4 ±t 1.9 N + CH N 1486 ± 141 CH N 1594 ± 115 After removal of old medium, the cells were washed two times with Hanks' balanced salts solution and incubated in 3 ml of Ham's F-1 at 37. Norepinephrine (N) was present at.1 mm and cycloheimide (CH) at 25 pg/ml. At the end of the first 4-hr incubation, the medium was removed, the cells were washed once, and 3 ml of medium containing the indicated drug were added for a second 1-min incubation. Cyclic AMP and phosphodiesterase were determined as described under Materials and Methods. The results represent the mean ±t SM (n = 4 for phosphodiesterase; n = 3 for cyclic AMP). The eperiment was repeated three times.
higher than control at the end of the 4 hr. A second addition of N caused only a 4.6-fold rise of camp, rather than the 14- fold elevation seen with the first N treatment. The presence of cycloheimide during the first 4 hr had no effect on the camp concentration when added either in the presence or absence of N. However, when cycloheimide had been present with N during the first incubation, a second addition of N now produced levels of camp comparable to those seen with a single N treatment. Since phosphodiesterase activity is at control level under these conditions, the results suggest that camp phosphodiesterase induction may cause at least partial refractoriness to a second N treatment. DSCUSSON The intracellular level of camp is determined by at least three factors: the rate of synthesis, the rate of degradation, and the rate of ecretion. Much work has shown that the rate of synthesis is affected by hormonal activation of the adenylate cyclase system. The etra camp produced is then degraded by the cyclic nucleotide phosphodiesterase, or partially ecreted in certain situations. This rise and fall of camp usually occurs within minutes, and is considered to be a shortterm response. Whereas the catecholamines and polypeptide hormones cause these short-term effects, other hormones, notably the steroids, can have more long-term effects on the basal level and/or responsiveness of the adenylate cyclase (14, 15). The results presented in this paper suggest that long-term hormonal regulation of the phosphodiesterase also eists. Treatment of the C-6 glioma cells with either Bt2cAMP or N resulted in increased phosphodiesterase activity. Since this increase was blocked by either cycloheimide or actinomycin D, it is the result of new RNA and protein synthesis. The simplest interpretation would be that additional molecules of the phosphodiesterase have been synthesized. However, it is also possible that the rate of synthesis of some effector protein, for eample, the heat-stable activator first described by Cheung (16), was increased. Further work will be required to distinguish between these possibilities. levated camp levels mediate phosphodiesterase induction in other cell lines. Dibutyryl-cAMP treatment increases phosphodiesterase activity, presumably by elevating intracellular camp levels, in several cell lines (3-6). Uzunov et al. (17) reported that norepinephrine increased one of two forms of phosphodiesterase in the C-2A astrocytoma cells. That camp eerts this effect via activation of the protein kinase is suggested by the work of Bourne et al. (6). They have isolated a lymphoma cell line deficient in protein kinase, in which the phosphodiesterase is no longer inducible by treatment with camp. posure to insulin has also been shown to result in increased phosphodiesterase activity in adipose tissue (18, 19), liver (2), and muscle (21), but these effects cannot be mediated by elevated camp levels since insulin in general lowers camp (22-24). Thus, there must eist at least two separate mechanisms whereby phosphodiesterase activity can be elevated. n the brain, increased neuronal activity may result in locally elevated concentrations of N. The short-term response of glia in the region might be elevation of camp levels, while the long-term response would be induction of phosphodiesterase activity. The phenomenon of refractoriness, whereby cells fail to respond to a second application of hor- 2 1 d 1 nduction of Cyclic AMP Phosphodiesterase 3847 1 2 3 4 5 TM (min) WTH N FG. 3. Correlation between intracellular cyclic AMP levels and low Km phosphodiesterase (PD) induction. The cells were grown as described under Materials and Methods. On the day of the eperiment, the cells were washed two times with 3-ml aliquots' of Hanks' balanced salts solution and incubated with 3 ml of Ham's F-1 containing.1 mm norepinephrine. For measurement of cyclic AMP, the medium was removed and the cells were fied in 1 ml of 5% trichloroacetic acid at the times indicated on the figure. Cyclic AMP was determined on the neutralized etracts as described under Materials and Methods. For phosphodiesterase activity, the medium was removed at the times indicated and the cells were washed once with 3 ml of medium and incubated with 3 ml of medium for 3 hr at 37. Phosphodiesterase activity was then determined on the suspended cells as described under Materials and Methods. ach point represents the mean of three dishes for cyclic AMP and four dishes for phosphodiesterase. The eperiment was repeated twice. mone when given within a short time of the first, has been demonstrated in this cell line for N and the camp response (8). The results in this paper suggest that at least a part of this refractoriness may be the result of the phosphodiesterase induction. Thus, phosphodiesterase induction may be one long-term response involved in control of repeated or ecess catecholamine stimulation. Since phosphodiesterase induction did not result in a complete refractoriness, however, other factors may also be involved. One possibility would be a desensitization of the adenylate cyclase (25) and a recent report presents evidence that such a mechanism occurs in a human astrocytoma cell line (26). These cells also become refractory to a second N stimulation. Although no change in phosphodiesterase was observed, the results suggested that there was a change in the responsiveness of the adenylate cyclase. 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