Journal of Neuroscience Research 2981-86 (1990)

Regulation of cAMP Levels by Protein Kinase C in C6 Rat Glioma Cells J. P. Bressler and P. Tinsely Surgical Neurology Branch and Laboratory of Neurobiology , National Institute of Health. Bethesda, Maryland

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13-dibutyrate (PDbU). An IC50 for PDbU of 38+5 nM and 62+8 nM was observed in the isoproterenol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproterenol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181-186,1987). An IC50 for phospholipase C of 6.020.1 X 10 -' and 7.0+0.1 X lo-' unitdm1 was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU-mediated inhibition to beta-adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta-adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed. Key words: glioma, CAMP,phosphodiesterase, phorbol esters, protein kinase C, differentiation INTRODUCTION Various types of biological phenomena have been shown to be activated after phorbol ester tumor promoter treatment (Blumberg, 1980). Phorbol esters may exert its effect by activating protein kinase C (Castagna et al., 1982), which in turn may directly effect gene activity, or it may indirectly effect gene activity by modulating levels of other second messengers (Bell et al, 1985; Brostrom et al., 1982). On the other hand, phorbol ester tumor promoters may mediate its effect through a protein 0 1990 Wiley-Liss, Inc.

kinase C independent mechanism. For example, the effects induced by phorbol esters are not always duplicated by permeant diacylglycerols, these include HL-60 differentiation (Kreutter et al., 1985), platelet activation (Ashby et al., 1985), and regulating phosphotidylcholine metabolism (Kolesnick and Paley, 1987). Phorbol ester tumor promoters have been shown to augment isoproterenol mediated increases in cAMP levels (Krdft and Anderson, 1987; Bell et al., 1985), while it has also been shown to suppress isoproterenol responses in other cell types (Garter and Belman, 1980; Brostrom et al., 1982). In this study we have examined suppression in the C6 rat gliorna cell line by asking two questions: 1) Does the elevation of diacylglcyerol levels effect the ability to increase cAMP levels similarly to phorbol ester tumor promoters? and 2) which mechanisms controling cAMP levels are effected by phorbol ester tumor promoter stimulation? This study was conducted in live cells, as opposed to membrane preparations, in order that some of the mechanisms controling cAMP levels, for example phosphodiesterases, be kept intact.

MATERIALS AND METHODS Cell Culture and Treatments All chemicals were of reagent grade and purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. The C6 2B subclone of the C6 cell line was cultured as described previously (de Vellis and Inglish,

Receivcd April 2.5, 1989; revised July 23, 1989; accepted August 5 . 1989. Address reprint requests to Joseph Bressler, Ph.D., Department of Neurology, The Kennedy Institute, 707 N . Broadway, Baltimore, MD 21205. Abbreviations used: CAMP = cyclic adenonsine 3'-S', monophosphate; GPDH = glycerol phosphate dehydrogenase; PDbU = phorbol 12, 13-dibutyrate; TCA = trichlororacetic acid; IBMX = 3-isobutylI-methyl-xanthine; ATP = adenosine 5'-triphosphate; BES-N,N-bis(2-hydroxyethy)-2-amino ethanesulfonic acid.

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1973; Bressler et al., 198.5). Cells were used 7-8 days after subculture. Phospholipase C (Clostridiurn wetchii), phospholipase A2 (EC 3.1.1.4, Crotulius adurnanteus), phospholipase D (EC 3.1.4.4, peanut from Calbiochem) were made fresh from a stock solution in media, diluted in media with 2% fetal bovine serum, and sterilized by filtration before adding to the cells.

cAMP Assays CAMP was assayed according to published procedures (Shimuzi et al., 1969) in triplicate. Cells were washed with serum-free media and incubated with I pCi of ['Hladenine per well (ICN, 40 Ciimmol) and the appropriate drug in serum-free media at 37°C. Unless stated otherwise, the cells were washed again and then challenged with isoproterenol (10pM) or forskolin (SOpM). After 1.5 min at 37"C, the cells were placed on an ice bath, washed with phosphate buffered saline, and the cAMP was extracted by treating the cells with 5% TCA. The TCA extract was applied to a Dowex-SOW-X4 column, which was followed by a series of elutions as described previously (Salomon et al., 1974). The water eluant was applied directly to a neutral alumina column, washed with water and eluted with 0.1 M imidazole, pH 7.2. Radioactivity was determined by liquid scintillation spectroscopy. Recovery was determined by the use of ['4C]cAMP (ICN, 40 Ci/mmol) as an internal standard (Wu and de Vellis, 1983). All values will be reported as the "percent conversion," which was computed by dividing the counts per minute of ['HICAMP X 100 by the counts per minute of [3H]ATP + [3H]cAMP. This method did not always allow us to detect the basal CAMP levels. Determining the Rate of cAMP Degradation The rate of loss of cAMP was assayed in intact cells by first prelabeling with ['Hladenine for 60 min and then stimulating the cells with isoproterenol (10pM) and IBMX (0.5 mM). After 30 min, cultures were washed five times with media containing 0. I % fatty-acid-free bovine serum albumin. Cultures were then treated with or without PDbU (83nM) in media, and at various time points the amount CAMP remaining in the cultures or in the supernatants was determined. Phosphodiesterase Assay Phosphodiesterase enzymatic activity in cell lysates was assayed by a previously reported procedure (Thompson et al., 1979) with modifications. Cells were grown as described as above and after 7 days were washed with BES buffered saline, pH 7.4. The cells were scraped into a BES buffer, pH 7.4, containing ovalbumin, 1 mgiml, and 1 mM EGTA and homogenized with a Brinkman Polytron. Phosphodiesterase enzymatic activity in 40-60

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Fig. 1. PDbU inhibits the beta-adrenergic and forskokin mediated increase in cAMP conversion. Seven-day-old cultures were washed with serum-free media and treated for 2 hr with (3H]adenine (1 p.Ci/ml). In the last hour various concentrations of PDbU were added. The cells were then challenged with isoproterenol (10 FM) (closed triangles) or forskoliri (50 FM) (open triangles) for 15 mins, after which the percentage of [3H]cAMP converted from ['HH]ATP was determined in the 5 % TCA extracts. The data is representative of four experiments.

pg of protein was assayed in 300 pl of a reaction mixture which contained 1 mM CAMP, 0.5 $1 ['HICAMP (ICN, 1.5 Ci/mmol), 1 mM MgCI,, 0.1 mM EGTA, and 1 mg/ml ovalbumin in BES buffer, pH 7.4. After a 30 min incubation at 30°C, the reaction was terminated by boiling. The tubes were then placed in an ice bath; and cobra venom, 50 p g per reaction, was added and the tubes were transferred to a 30°C water bath for 30 min. The tubes were again placed in an ice bath and Dowex acetate was added to each tube and the amount of radioactivity which failed to bind to the resin was determined. Protein was determined by the method of Lowry et a]., (19.51).

RESULTS PDbU Inhibits the Beta-Adrenergic and Forskolin Response The isoproterenol response or forskolin response was examined in 7-day-old C6 rat glioma cell cultures that were labeled with ['Hladenine for 2 hr and treated in the last hour with PDbU. An inhibition in both responses was observed in PDbU treated cells (Fig. 1). In four different experiments, an average IC.50 for PDbU of 3 8 2 5 nM or 6 2 k 8 nM was observed in the isoproterenol and forskolin response, respectively. Nontreated cells exhibited a response of 1.70+.06% and 2.5+0.6%, for the isoproterenol and forskolin treatment, respectively. Very high concentrations of PDbU (over 500 nM) did not reduce the isoproterenol or the forskolin response to

Regulation of cAMP Levels by Protein Kinase C

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Fig. 2. Phospholipase C (triangles),but not phospholipase A2 (squares) or D (circles) inhibits the isoproterenol (10 p M ) (closed symbols) and forskolin (SO pM) (open symbols) mediated increase in cAMP conversion. Six-day-oldcultures were washed and treated for 16 hr with various concentrations of enzymes. The cells were labeled with ['Hladenine (1 pCi/ml) for 2 hr and the percentage of ['HICAMP converted from ['HIATP in isoproterenol (closed symbols) or forskokin (open symbols) treated cells was determined. The data are representative of three experiments. basal levels. PDbU was found not to have an effect on the total incorporation of ['Hladenine or on levels of ['HIATP (data not shown).

Phospholipase C Inhibits the Isoproterenol and Forskolin Response Cultures treated with phospholipase C exhibit higher diacylglycerol levels which bind to the phorbol ester receptor (Bressler, 1987). Cultures treated with this enzyme overnight and labeled with ['H]adenine for 2 hr also have a diminished response to isoproterenol and forskolin (Fig. 2). An average IC50 of 6.020. I X lop2 and 7.020.1 X lo-' unitdm1 was observed for the isoproterenol and forskolin response, respectively. The isoproterenol and forskolin response in nontreated cells was 1.9+0.05% and 2.6+0.15%, respectively. No change in the ability of isoproterenol or forskolin to increase cAMP levels was observed for cells treated with phospholipase A2 or phospholipase D even at concentrations as high as 1 unit/ml. This suggests that the inhibition mediated by phospholipase C was not due to a nonspecific membrane perturbation. Kinetics of PMA-Mediated Inhibition The kinetics of the PDbU-mediated inhibition of the forskolin and isoproterenol response was investigated in order to determine whether PDbU inhibits these responses at the same site. We reasoned that if two sites were affected (for example, the beta-adrenergic receptor

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Fig. 3 . A kinetic study of the PDbU mediated inhibition of the forskokin and beta-adrenergic response. Seven-day-old C6 cultures were washed with serum-free media and labeled with [3H]adenine(1 pCi/ml). At sequential time points during this labeling period, cultures were treated with PDbU (83 nM). After 2 hr, cultures were washed, stimulated with either isoproterenol (10 pM) for 15 min or forskolin (SO pM) for IS mins, and the percentage of ['HICAMP converted was determined. The data are expressed as time versus the percent inhibition of the forskolin (closed triangles) or isoproterenol (open triangles) response. An 100% response is the isoprotereno1 or forskolin response without PDbU. The data are representative of four experiments. and a GTP-binding protein) then the minimum time needed to see a PDbU mediated inhibition in the isoproterenol and forskolin might also be different. Cultures were washed and labeled with ['H]adenine for 2 hr. Cultures were preincubated with PDbU (83 nM) for various time points during the labeling period and were evaluated for their ability to convert ATP to cAMP after forskolin or isoproterenol stimulation. In three different experiments, a small but significant inhibition in the isoproterenol response occurred earlier than the inhibition of the forskolin response. In the experiment shown in Figure 3, a 30 min incubation induced an 18% inhibition in the isoproterenol response ( P c0.05). No change in the forskolin response was observed. By 1 hr the degree of PDbU mediated inhibition in the forskolin and isoproterenol response was similar. By 2 hr the maximum degree of inhibition in both responses was obtained. These differences in the kinetics suggested that the mechanisms whereby PDbU influences the forskolin and isoprotereno1 may be different.

Forskolin Dose Response Studies were initiated to determine the site(s) where PDbU inhibited the forskolin mediated increase in cAMP levels. Forskolin is capable of increasing cAMP levels in most cell types by directly activating adenylate

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FIg. 4. Forskolin dose-response curve on PDbU treated cells. Seven-day-oldC6 cultures were washed with serum-free media and treated with either PDbU (83 nM) (open triangles) in media containing ['Hladenine ( I pCiiml) or with labeling media devoid of PDbU (open circles). Half the cultures were treated with PDbU for the last hour. Cells were stimulated with various concentrations of forskolin and the percentage of 13H]cAMPconverted from ['HIATP was determined. The data are representative of three experiments.

high end of a dose response curve would suggest a possible site. C6 glioma cultures were labeled, treated with PDbU at 83 nM, and stimulated with forskolin for 15 min. In three different experiments, we observed that the degree of PDbU mediated inhibition was similar at all of the forskolin concentrations (Fig. 4).

The Effect of PDbU on Phosphodiesterase Activity A possible site where cells can modulate cAMP levels is by altering its rate of degradation. Therefore, the possibility that PDbU was stimulating cAMP degradation in intact cells was investigated. Cultures were labeled for 2 hr with ['Hladenine and then were stimulated for 30 min with isoproterenol (10 p,M) and IBMX (0.5 mM). The cells were washed and treated with either medium or medium containing PDbU at 83 nM. In four different experiments, cAMP levels were found to decrease faster in cultures treated with PDbU (Fig. 5 ) . In the experiment shown in Figure 5 , cells treated with PDbU for 55 min exhibited 20% (a rate of conversion of 0.1% as compared to 0.5%) of the response of nontreated cells. No change in the amount of cAMP released into the media was observed (data not shown). In three different experiments, we found a 5 1 2 9 % increase in phosphodiesterase activity in cultures treated for one hour with PDbU at 83 nM.

DISCUSSION In this study we have demonstrated that, similar to PDbU treatment, phospholipase C treated C6 rat glioma cells have a diminished capability to increase cAMP lev0.5 els in response to isoproterenol and forskolin. These observations are comparable to those made in previous experiments in which we demonstrated that cells treated with phospholipase C exhibited elevated diacylglycerol 15 30 45 60 75 levels, exhibited a higher K, for PDbU and similar to TIME lminutes) phorbol ester treated cells, were inhibited from increasFig. 5 . A kinetic study of the effect of PDbU on the disaping GPDH levels after glucocorticoid stimulation pearance of induced levels of CAMP. Seven-day-old cultures (Bressler, 1987). Therefore the PDbU mediated inhibiwere washed with serum-free media and labeled with [3H]adenine(1 pCi/ml). After 2 hr cultures were treated with tion of the beta-adrenergic response in C6 rat glioma isoproterenol (10 kM) and IBMX (0.5 mM) for an additional cells was probably due to a direct involvement by the half-hour, washed with media containing 0.1 % fatty acid free protein kinase C. BSA, and the cultures were fed with either media (open circles) Previous work by Kassis et al. ( 1 985) using broken or PDbU at 83 nM (closed circles). At subsequent time points cell preparations, demonstrated that phorbol esters inthe rate of conversion in either the cells or in the media was hibit the beta-adrenergic response in C6 cells by causing determined. an heterologous desensitization of the beta-adrenergic receptor. N o change in Gi, Gs, or the catalytic component of adenylate cyclase was observed. This would not excyclase as well as stimulating the Gs protein (Daly, plain why in our experiments the forskolin response is 1984). Adenylate cyclase and the Gs protein most likely inhibited since forskolin does not effect the beta-adrenare the low- and high-affinity forskolin binding sites, ergic receptor, but rather, in a concentration dependent respectively, that have been described (Nelson and Sea- manner, increases cAMP at Ns or at the adenylate cymon, 1986). We reasoned that inhibition at the low or clase (Daly, 1984). Our data suggests that another mecht

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Regulation of cAMP Levels by Protein Kinase C

anism is being activated by PDbU, namely, an increase in phosphodiesterase activity. In human glioma cells, diminished cAMP levels was also shown to be caused by an activated phosphodiesterase after muscarinic cholinergic stimulation (Tanner et al., 1986). In this example, a calmodulin-dependent phosphodiesterase was activated within minutes of cholinergic stimulation, which is in contrast to our work where a phosphodiesterase is activated after a 45 min incubation. In contrast, phosphodiesterase activity was shown to be inhibited by phorbol esters in anterior pituitary cell cultures (Abou-Samra et a]., 1987). A simple mechanism that might help explain the increase in phosphodiesterase activity is that the enzyme activity can be regulated by protein kinase C induced phosphorylation. Several types of phosphodiesterases have been reported (Strada et al., 1984), some of which are activated by phosphorylation. The cGMP-inhibited phosphodiesterase of human platlets (Macphee et al., 1988) and a low K,, phosphodiesterase of rat adipocytes (Gettys et al., 1988) have both been shown to be activated by CAMP-dependent protein kinase. These phosphodiesterases may also serve as a protein kinase C substrate since the same protein may serve as a substrate for two different types of protein kinases, though the site of phosphorylation will be different. For example, a calmodulin-dependent phosphodiesterase has been shown to serve as a substrate for a calmodulin-dependent protein kinase, as well as for a CAMP-dependent protein kinase (Sharma and Wang 1985, 1968). In addition, another enzyme, tyrosine hydroxylase, can be phosphorylated by a CAMP-dependent protein kinase (Joh et al., 1978), a calmodulin-dependent mechanism (Yamauchi et al., 1981; Vulliet, 1984) and the protein kinase C (Pocotte et al., 1986). In conclusion, activation of protein kinase C, in intact C6 glioma cells, leads to the inhibition of the betaadrenergic and forskolin mediated increase in cAMP levels. This inhibition was probably due to both a desensitization of the beta-adrenergic receptor (Kassis, 1985) as well as activation of phosphodiesterase. There may have also been other sites affected by protein kinase C but which we were unable to resolve in our system. The inability of these cells to maintain cAMP levels under these conditions may help explain why phorbol esters and phospholipase C treated C6 glioma cells have a diminished capacity to elevate GPDH levels after glucocorticoid stimulation (Bressler et al., 1985; Bressler, 1987), since cAMP serves as a constituent component in GPDH induction (Breen et al., 1978). We would also like to point out that sodium butyrdte at concentrations above 200 pM (which is well above the concentration of PDbU used in these studies) acts as an inhibitor of GPDH (Weingarten and de Vellis, 1980) and S-100 induction,

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and inhibits the beta-adrenergic and forskokin response in C6 cells (Hirshfeld and Bressler, 1987). Therefore, it appears that maintenance of adequate cAMP levels may be crucial in expressing differentiated properties in C6 rat glioma cells.

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Sharma, RK, Wang, JH (1986): Calmodulin and Ca2 -dependent phosphorylation and desphosphorylation of 63-kDA subunitcontaining bovine brain calniodulin-stimulated cyclic nucleotide phosphodieterase isozyme. J Biol Chem 261: 1322-1 328. Shimizu, H, Daly, J , Creveling, CR (1969). A radioisotopic method for measuring the formation of adenosine 3' .S'-cyclic monophsophate in incubated slices of rat brain. J Neurochem 16: 1609-1 619. Strada, SJ, Martin, MW, Thompson, WJ (1984): General properties of multiple molecular forms of cyclic nucleotide phosphodiesterase in the nervous system. Adv Cyclic Nucl Protein Phosphor Ues 1613-29. Tanner, L, Harden, TK, Wells, JN, Martin, MW (1986): Indirect identification of the phosphodiesterase isozyme regulated by muscarinic receptors in astrocytoma cells. Mol Pharmacol 29: 455-460. Thompson, WJ, Terasaki, WL, Epstein, PM, Strada, SJ (1979): Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme. Adv Cyclic Nucleotide Res 10:69-92. Vulliet, PR, Woodgut, JR, Cohen, P (1984): Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase. J Biol Chem 259:13680-13683. Weingarten, D, de Vellis, J (1980): Selective inhibition by sodium butyrate of the glucocorticoid induction of glycerol phosphate dehydrogenase in glial cultures. FEBS Letters 126:289-291. Wu, DK, de Vellis, J (1983): Effect of forskolin on primary cultures of astrocytes and oligodendrocytes. J Cyclic Nucleotide Protein Phosphor Res 9159-67. Yamauchi, T, Nakata, H, Fujisawa, H (1981): A new activator protein that activate tryptophan S-monooxygenase in the presence of Ca', calmodulin-protein kinase. J Biol Chem 2565404-5409,