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Protein kinase C is involved in cholecystokinin octapeptide-induced proliferative action in rat glioma C6 cells
Neuropeptides(1998) 32 (2), 185-189 © HarcourtBrace and Co. Ltd 1998
Protein k i n a s e C is involved in Cholecystokinin Octapeptide.induced proliferative a c t i o n in rat glioma C 6 cells R. K a u f m a n n , 1 H. S c h a f b e r g , 1 M. Zieger, 1 P. H e n k l e i n , 2 G. N o w a k 1 ~Research Unit, 'Pharmacological Haemostaseology', Medical Faculty at the Friedrich-Schiller-University Jena, Drackendorfer Str. 1, 07747 Jena 2institute of Biochemistry, Medical Faculty (Charit6) at the Humboldt University, Berlin, Germany
Summary Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C 6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C 6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma Ce cells with CCK-8S resulted in activation of protein kinase C isozymes ]31,1311,7 and 4. The participation of protein kinase C in the CCK-8Sinduced effect on C 6 cell growth was demonstrated by measurement of [3H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C 6 cells by a protein kinase C-dependent mechanism.
INTRODUCTION
Cholecystokinin (CCK), originally identified as a gastrointestinal polypeptide, ~ is widely distributed throughout the central nervous system (CNS) where it has been suggested to play a role as neurotransmitter or neuromodulator? The predominant form of neuronal CCK is CCK-SS, a sulphated carboxyterminal octapeptide2, 4 Receptors for CCK in both the CNS and peripheral tissues can be classified into at least two types, CCKA and CCKB, 5 of which CCKB sites are the predominant form in the brain. A recent study characterized high affinity CCKB binding sites on rat glioma Ca cells 6 and demonstrated CCKB receptor-mediated intracellular signalling 7 and stimulation of DNA synthesis in C a cells. 8 This study reports that stimulation of rat glioma C a cells with CCK-SS results in activation of protein kinase C (PKC) isoforms ~I, ~II, 7 and ~ and demonstrates the
involvement of protein kinase C in cholecystokinininduced growth action in C a cells.
MATERIALS AND METHODS Materials
Sulphated CCK-8 (CCK-8S) was synthesized as described previously? Further compounds were obtained from the following sources: [methyl-3H]thymidine (70-85 Ci/ mmol; Amersham Buchler GmbH, Germany), phorbol- 12myristate-13-acetate (PMA) and 2-[1-(3-dimethylaminopropyl)- 1H-indol-3-yl]-3-(1 H - i n d o l - 3 - y l ) - m a l e i m i d e (bisindolylmaleimide I, BIM) (Calbiochem). The rat glioma cell line C a was from the American Type Culture Collection (ATCC, USA). Cell culture
Received 15 September 1997 Accepted 17 December 1997
Correspondence to: R. Kaufmann, Research Unit, 'Pharmacological Haemostaseelogy', Medical Faculty at the Friedrich Schiller University Jena, Drackendorfer Str. 1,07747 Jena, Germany. Tel: +49-3641-304427, Fax: +49-3641-304412, e-mail:rol @ leech.mpg.uni-jena.de
Rat glioma C 6 cells were grown as monolayer cultures in Ham's F-12 medium supplemented with 2.5% fetal calf serum and 150/0 horse serum in cell culture flasks. Cells were routinely cultured at 37°C in a humidified atmosphere. The gas phase was 95% air and 5% CO 2. The culture medium was changed every 2-3 days. 185
186
Kaufmann et al
Membrane preparation
C a cells were grown in six-well plates for 24 h. Cells were then exposed to CCK-8S for 5 rain at 37°C. The incubation was terminated by removal of the cells and centrifugation at 20 000 x gfor 3 rain (4°C). The sedimented cells were washed with 2 ml of 50 rnM Hepes, pH Z4, and recentrifuged at 100 000 × g for 60rain (4°C). The cell pellets were resuspended in 50 mlVI Hepes, pH Z4, containing bacitracin (100 ~tg/rnl), PMSF (0.1 raM), pepstatin .A (1 ~tg/ml) and leupeptin (2 ~tg/rnl) and stored at -80°C. Protein was determined according to Bradford ~°with BS.A as standard. Preparation of whole cell lysates
C a cells were collected by centrifugation at 1000 x gfor 5 min (4°C), washed with PBS containing bacitracin (100 ~tg/ml), PMSF (0.1 raM), pepstatin .A (1 ~tg/ml) and leupeptin (2 ~g/ml), pH 7.4, and centrifuged again. The pellet was treated with lysis buffer (PBS, containing 1% v/v Triton X-100, 0.5% w/v deoxycholate and 0.1% w/v SDS for 30 rain at 4°C, resuspended and centrifuged at 30 000 x g for 15 rain (4°C). The supernate was mixed with SDS/P.AGE buffer and boiled for 5 rain. Determination of PKC isoforms and PKC activation
Western blot analysis of PKC was performed with polyclonal anti-PKC cz, [3I, [~II,7, 8, e, 4, 11, ~, 0 antibodies (Santa Cruz). Proteins of rat glioma C a cell lysates or membranes were separated on 10% SDS/P.AGE and transferred to nitrocellulose membranes (BioRad). After blocking nonspecific immunolabelling sites on the nitrocellulose in 1% BS.A/I% non-fat dried milk powder for I h the nitrocellulose strips were incubated overnight with the various anti-PKC antibodies (1.0 gg/ml blocking solution). After appropriate washing with Tween 20 (0.05% v/v) washing buffer, the blots were incubated for 45 min with goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz) and washed again twice as described above. Secondary antibodies were detected by a chemiluminescence (ECL) Western blotting detection system (Amersham) by exposure to Kodak X-Omat films. [3H]thymidine assay
C a cells were grown in 75 cm 2 cell culture flasks..At confluency cells were passaged into 12-well plates with Ham's F-12 medium containing 15% horse serum and 2.5% FCS for 24h. After incubation in serum-free medium for 12 h, the cells were stimulated with the different experimental reagents and labelled with [methyl3H]thymidine (5.0 lxCi/well) for 24 h. Then the cells were kept on ice and trichloroacetic acid (10%) was added for Neuropeptides (1998) 32(2), 185-189
15 man on ice and rinsed twice with ice-cold H20. After treatment with ice-cold methanol, the cells were lysed with NaOH and incorporated [3HI was counted by liquid scintillation (Wallac 1410 Liquid Scintillation Counter). Protein was measured as described previously? ~ Cell growth assay
The C a cell growth assay was performed as described previouslyY Briefly, C a glioma cells were grown on CELLocate coverslips (alphabetically labelled squares, square size 175 mm, Eppendorf) placed in six-well plates in Ham's-F12 medium containing 15% horse serum and 2.5% FCS for 48h. After incubation in serum-free medium for 24 h, a baseline cell count was performed with an Axiovert 135 microscope (Carl Zeiss). Then, the cells were treated for a further 48 h with test agents. After this period, the cells were counted again and the increase in cell number was determined. Cell viability was estimated by trypan blue exclusion assay. RESULTS
Western blotting of whole cell extracts established that rat glioma C 6 cells contain the protein kinase C isoforms cz, [3I, [~II,7, 8, ~ and ~ (Fig. 1) whereas the PKC subspecies q, t and 0 do not occur in significant amounts (data not shown). The immunoreactivity of PKC subspecies could be blocked by incubation with the respective antigenic peptide (data not shown). Translocation of conventional, novel and atypical PKC isoforms from cytosol to the plasma membrane (increased association with the particulate fraction) results in their activation. 13,~4Stimulation of C 6 cells with CCK-8S (final concentration 10 niVl) yielded translocation of PKC isoforms [~I,[~II,7 and ~ to the cell membrane. This effect was observed 1-12 min after treatment of C a cells with CCK-SS and is shown in Figure 2 for C a cells stimulated with CCK-8S for 5 min. After longer stimulation (15, 20, 30 and 60 min) no more PKC activation could be seen (data not shown). In addition, no increased association of the isoforms c~, 8 and ~ with the particulate fraction of C 6 cells could be observed (Fig. 2). Next, the effect of CCK-8S on C 6 cell growth was investigated by measurement of [3H]thymidine incorporation and estimation of cell number. As shown in Figure 3, CCK-8S (final concentration 10 nM) induced significant enhancement of [3H]thymidine uptake in rat glioma C 6 cells. This effect could be prevented when C a cells were preincubated with the PKC inhibitor BIM (1.0 ~M) for 30 man. In addition, the phorbolester PM_A (final concentration 1.0 ~tM) also induced increase in [3H]thymidine uptake in Ca glioma cells (Fig. 3). As further shown in Figure 3, no © Harcourt Brace and Co. Ltd 1998
CCK-8S-induced proliferative action
187
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Fig. 1 PKC subspecies 0~, ~1, ~ll, ~', 5, s and ( are present in rat glioma C 6 cells. C 6 cell lysate was separated by 10% SDS-PAGE and transferred to nitrocellulose strips. PKC subspecies were estimated as described in Materials and methods. Migration of molecular mass marker (90 kDa, Santa Cruz) is indicated on the left.
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CCK-8S induces activation of the PKC isoforms ~1, I~11,y and ( but not o~, 8 and s. C 6 cell membranes were prepared as described ~n
Materials and methods. The membranes were analysed by immunoblotting with polyclonal antibodies against the respective subspecies of PKC (1 #g/ml). Migration of molecular mass marker (90 kDa) is indicated on the left. -, non-stimulated MIA-PaCA-2 cells; +, C 6 cells cells stimulated with CCK-8S (10 riM)
additional increase in [3H]thymidine incorporation was observed after simultaneous stimulation of C 6 glioma cells with PMA (final concentration 1.0 gM) and CCK-SS (final concentration 10 nM) compared with the effect of PMA alone. When C6 cells were exposed to CCK-8S (final concentration 10 nM) significantly enhanced cell numbers could be noted in comparison with non-treated C 6 cells (Fig. 4). This effect could be mimicked by the phorbolester PMA (final concentration 1.0 gM) and totally blocked when Ca cells were preincubated with the PKC inhibitor BIM (1.0 ~tM) for 20 rain (Fig. 4).
© Harcourt Brace and Co. Ltd 1998
DISCUSSION Earlier studies have described high affinity CCKB-type receptors and their connection with inositol metabolism, 7 calcium signalling6, 7 and protein phosphorylation7 CCK8S-induced increase in [3H]thymidine incorporation in rat glioma Ca was also reported. 8 However, the involvement of protein kinase C activation in CCK action on C6 cells has not been demonstrated to date. It is well known that receptors that use calcium as a second messenger hydrolyse phosphoinositides resulting Neuropeptides (1998) 32(2), 185-189
188 Kaufmann et al
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in generation of InsP 3 and diacylgycerol. The InsP 3 release in the cytoplasm mobilizes calcium from internal stores, whereas diacylglycerol activates protein ldnase C. ~5 Members of the PKC family play a key regulatory role in a variety of cellular functions, including cell growth and differentiation. 1¢17 The purpose of the present study was to investigate the involvement of PKC in CCK-8S-induced growth effects in rat glioma C a cells. When C a cells were exposed to CCK8S activation of b o t h the conventional PKC isoforms [~I, ~II, 7 requiring calcium and diacylglycerol, and the atypical isoform ~, being b o t h calcium and diacylglycerol independent could be observed. It is known that the mitogenic effect of growth factors and phorbolesters m a y be mediated through PKCY ,2° Because CCK-8S-induced increase in [3H]thymidine incorporation and cell n u m b e r could be blocked by the PKC inhibitor bisindolylmaleimide, the involvement of protein ldnase C in CCKmediated growth action in C a glioma cells is highly indicated. This was supported by the findings that the phorbolester PMA mimicked the CCK-8S-induced effect on C a cell growth and no additive effect of CCK-SS on PMA-induced [3H]thymidine incorporation could be observed in C a glioma. To summarize, the involvement of protein kinase C in cholecystokinin-induced proliferative action in rat glioma C a cells has been demonstrated. Further investigations in this field are in progress, including b o t h the role of the individual PKC subspecies and their connecNeuropeptides (1998) 32(2), 185-189
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Fig. 3 [3H]thymidine incorporation in rat glioma Cs cells exposed to different experimental reagents. Cells were loaded with [methyl3H]thymidine as described in Materials and methods and radioactivity was counted by liquid scintillation. Control: [~H]thymidine incorporation in non-stimulated Ce cells. Values are the means _+SE of three independent experiments, each done in triplicate.
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Fig. 4 Effect of CCK-8S, PMA and bisindolylmaleimide I on the proliferation of C6 glioma cells. Experiments were performed as described in Materials and methods. Values represent the mean + SE percentage increase in cell number of at least three independent experiments, each performed in duplicate. Control: non-stimulated C6 cells. Data are presented as percentage of control (cell number = 467 + 21).
tion with signalling events downstream of the protein kinase C, including Raf and the MAP kinase pathway. ACKNOWLEDGEMENT
This study was supported partly by Grant II B6-Ka 1098/1-2 from the Deutsche Forschungsgemeinschaft (DFG). REFERENCES
1. Mutt V,Jorpes J E. Hormonal polypeptides of the upper intestine. EurJ Biochem 1971; 6: 156-162. 2. Vanderhaeghen J J, Lotslra F, De Mey J, Gilles C. Immunhistochemical location of cholecystokinin- and gastrinlike peptides in the brain and hypophysis of the rat. Proc Nail Acad Sci USA 1980; 77:1190-1194. 3. Dockray G J, Gregory R A, Hutchinson J B. Isolation, structure and biological activity of two cholecystoldnin octapeptides from sheep brain. Nature 1978; 274:711-713. 4. Rehfeld J F. Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogeneity in the central nervous system and small intestine of man and hog. J Biol Chem 1978; 253: 422-4030. 5. Moran T H, Robinson P H, Goldrich M S, McHugh P R. Two brain cholecystokinin receptors: implication for behavioral actions. Brain Res 1986; 362: 175-179. 6. Kaufmann R, Lindschau C, Sch6neberg T et al. Type B cholecystokinin receptors on rat glioma Cs cells. Binding studies and measurement of intracellular calcium mobilization. Brain Res 1994; 639:109-114. © Harcourt Brace and Co. Ltd 1998
CCK-8S-induced proliferative action
Z Kaufmann R, Lindschau C, Hailer H, Sch6neberg T, Ott 32 CCK8S induced signalling in rat glioma C6 cells. Neuropeptides 1995; 29: 251-256. 8. Kaufmann R, Henklein P, Nowak G. CCKs receptor activation is resulted in increased pH]thymidine incorporation in rat glioma C6 cells. Neuropeptides 1995; 29:241-244. 9. Henklein P, Penke B, Georgi M e t al. Verbesserte Synthese der hochaktiven Cholecystokininsequenz Succinyl-Tyr(OSO3)-MetGly-Trp-Asp-Phe-NH2. Pharmazie 1987; 42: 413-414. 10. Bradford M M. A refined and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254. 11. Lowry O H, Rosebrough N J, Farr A L, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275. 12. Schafberg H, Nowak G, Kaufmann R. Thrombin has a bimodal effect on glioma cell growth. Br J Cancer 1997 (in press).
© Harcourt Brace and Co. Ltd 1998
189
13. Mexander D R, Cantrell D A. Kinases and phosphatases in T-cell activation. Immunol Today 1989; 10: 200-205. 14. Huang K P. The mechanism of protein kinase C activation. Trends Neurosci 1989; 12: 425-432. 15. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promoting. Nature 1984; 308: 693-69Z 16. Clemens M J, Trayner I, Menaya J. The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J Clin Invest 1992; 103:881-88Z 1Z Rozengurt E. Neuropeptides as cellular growth factors: role of multiple signalling pathways. Eur J Clin Invest 1991; 21 : 123-134. 18. Dekker L V, Parker PJ. Protein kinase C - a question of specifity. Trends Biochem Sci 1994; 19: 7 3 - 7 Z 19. Hug H, Sarre T F. Protein kinase C isoenzymes: divergence in signal transduction. Biochem J 1993; 291: 329-343. 20. Stabel S, Parker P J. Protein kinase C. Pharmacol Ther 1991; 51 : 71-95.
Neuropeptides (1998) 32(2), 185-189
Protein k i n a s e C is involved in Cholecystokinin Octapeptide.induced proliferative a c t i o n in rat glioma C 6 cells R. K a u f m a n n , 1 H. S c h a f b e r g , 1 M. Zieger, 1 P. H e n k l e i n , 2 G. N o w a k 1 ~Research Unit, 'Pharmacological Haemostaseology', Medical Faculty at the Friedrich-Schiller-University Jena, Drackendorfer Str. 1, 07747 Jena 2institute of Biochemistry, Medical Faculty (Charit6) at the Humboldt University, Berlin, Germany
Summary Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C 6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C 6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma Ce cells with CCK-8S resulted in activation of protein kinase C isozymes ]31,1311,7 and 4. The participation of protein kinase C in the CCK-8Sinduced effect on C 6 cell growth was demonstrated by measurement of [3H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C 6 cells by a protein kinase C-dependent mechanism.
INTRODUCTION
Cholecystokinin (CCK), originally identified as a gastrointestinal polypeptide, ~ is widely distributed throughout the central nervous system (CNS) where it has been suggested to play a role as neurotransmitter or neuromodulator? The predominant form of neuronal CCK is CCK-SS, a sulphated carboxyterminal octapeptide2, 4 Receptors for CCK in both the CNS and peripheral tissues can be classified into at least two types, CCKA and CCKB, 5 of which CCKB sites are the predominant form in the brain. A recent study characterized high affinity CCKB binding sites on rat glioma Ca cells 6 and demonstrated CCKB receptor-mediated intracellular signalling 7 and stimulation of DNA synthesis in C a cells. 8 This study reports that stimulation of rat glioma C a cells with CCK-SS results in activation of protein kinase C (PKC) isoforms ~I, ~II, 7 and ~ and demonstrates the
involvement of protein kinase C in cholecystokinininduced growth action in C a cells.
MATERIALS AND METHODS Materials
Sulphated CCK-8 (CCK-8S) was synthesized as described previously? Further compounds were obtained from the following sources: [methyl-3H]thymidine (70-85 Ci/ mmol; Amersham Buchler GmbH, Germany), phorbol- 12myristate-13-acetate (PMA) and 2-[1-(3-dimethylaminopropyl)- 1H-indol-3-yl]-3-(1 H - i n d o l - 3 - y l ) - m a l e i m i d e (bisindolylmaleimide I, BIM) (Calbiochem). The rat glioma cell line C a was from the American Type Culture Collection (ATCC, USA). Cell culture
Received 15 September 1997 Accepted 17 December 1997
Correspondence to: R. Kaufmann, Research Unit, 'Pharmacological Haemostaseelogy', Medical Faculty at the Friedrich Schiller University Jena, Drackendorfer Str. 1,07747 Jena, Germany. Tel: +49-3641-304427, Fax: +49-3641-304412, e-mail:rol @ leech.mpg.uni-jena.de
Rat glioma C 6 cells were grown as monolayer cultures in Ham's F-12 medium supplemented with 2.5% fetal calf serum and 150/0 horse serum in cell culture flasks. Cells were routinely cultured at 37°C in a humidified atmosphere. The gas phase was 95% air and 5% CO 2. The culture medium was changed every 2-3 days. 185
186
Kaufmann et al
Membrane preparation
C a cells were grown in six-well plates for 24 h. Cells were then exposed to CCK-8S for 5 rain at 37°C. The incubation was terminated by removal of the cells and centrifugation at 20 000 x gfor 3 rain (4°C). The sedimented cells were washed with 2 ml of 50 rnM Hepes, pH Z4, and recentrifuged at 100 000 × g for 60rain (4°C). The cell pellets were resuspended in 50 mlVI Hepes, pH Z4, containing bacitracin (100 ~tg/rnl), PMSF (0.1 raM), pepstatin .A (1 ~tg/ml) and leupeptin (2 ~tg/rnl) and stored at -80°C. Protein was determined according to Bradford ~°with BS.A as standard. Preparation of whole cell lysates
C a cells were collected by centrifugation at 1000 x gfor 5 min (4°C), washed with PBS containing bacitracin (100 ~tg/ml), PMSF (0.1 raM), pepstatin .A (1 ~tg/ml) and leupeptin (2 ~g/ml), pH 7.4, and centrifuged again. The pellet was treated with lysis buffer (PBS, containing 1% v/v Triton X-100, 0.5% w/v deoxycholate and 0.1% w/v SDS for 30 rain at 4°C, resuspended and centrifuged at 30 000 x g for 15 rain (4°C). The supernate was mixed with SDS/P.AGE buffer and boiled for 5 rain. Determination of PKC isoforms and PKC activation
Western blot analysis of PKC was performed with polyclonal anti-PKC cz, [3I, [~II,7, 8, e, 4, 11, ~, 0 antibodies (Santa Cruz). Proteins of rat glioma C a cell lysates or membranes were separated on 10% SDS/P.AGE and transferred to nitrocellulose membranes (BioRad). After blocking nonspecific immunolabelling sites on the nitrocellulose in 1% BS.A/I% non-fat dried milk powder for I h the nitrocellulose strips were incubated overnight with the various anti-PKC antibodies (1.0 gg/ml blocking solution). After appropriate washing with Tween 20 (0.05% v/v) washing buffer, the blots were incubated for 45 min with goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz) and washed again twice as described above. Secondary antibodies were detected by a chemiluminescence (ECL) Western blotting detection system (Amersham) by exposure to Kodak X-Omat films. [3H]thymidine assay
C a cells were grown in 75 cm 2 cell culture flasks..At confluency cells were passaged into 12-well plates with Ham's F-12 medium containing 15% horse serum and 2.5% FCS for 24h. After incubation in serum-free medium for 12 h, the cells were stimulated with the different experimental reagents and labelled with [methyl3H]thymidine (5.0 lxCi/well) for 24 h. Then the cells were kept on ice and trichloroacetic acid (10%) was added for Neuropeptides (1998) 32(2), 185-189
15 man on ice and rinsed twice with ice-cold H20. After treatment with ice-cold methanol, the cells were lysed with NaOH and incorporated [3HI was counted by liquid scintillation (Wallac 1410 Liquid Scintillation Counter). Protein was measured as described previously? ~ Cell growth assay
The C a cell growth assay was performed as described previouslyY Briefly, C a glioma cells were grown on CELLocate coverslips (alphabetically labelled squares, square size 175 mm, Eppendorf) placed in six-well plates in Ham's-F12 medium containing 15% horse serum and 2.5% FCS for 48h. After incubation in serum-free medium for 24 h, a baseline cell count was performed with an Axiovert 135 microscope (Carl Zeiss). Then, the cells were treated for a further 48 h with test agents. After this period, the cells were counted again and the increase in cell number was determined. Cell viability was estimated by trypan blue exclusion assay. RESULTS
Western blotting of whole cell extracts established that rat glioma C 6 cells contain the protein kinase C isoforms cz, [3I, [~II,7, 8, ~ and ~ (Fig. 1) whereas the PKC subspecies q, t and 0 do not occur in significant amounts (data not shown). The immunoreactivity of PKC subspecies could be blocked by incubation with the respective antigenic peptide (data not shown). Translocation of conventional, novel and atypical PKC isoforms from cytosol to the plasma membrane (increased association with the particulate fraction) results in their activation. 13,~4Stimulation of C 6 cells with CCK-8S (final concentration 10 niVl) yielded translocation of PKC isoforms [~I,[~II,7 and ~ to the cell membrane. This effect was observed 1-12 min after treatment of C a cells with CCK-SS and is shown in Figure 2 for C a cells stimulated with CCK-8S for 5 min. After longer stimulation (15, 20, 30 and 60 min) no more PKC activation could be seen (data not shown). In addition, no increased association of the isoforms c~, 8 and ~ with the particulate fraction of C 6 cells could be observed (Fig. 2). Next, the effect of CCK-8S on C 6 cell growth was investigated by measurement of [3H]thymidine incorporation and estimation of cell number. As shown in Figure 3, CCK-8S (final concentration 10 nM) induced significant enhancement of [3H]thymidine uptake in rat glioma C 6 cells. This effect could be prevented when C a cells were preincubated with the PKC inhibitor BIM (1.0 ~M) for 30 man. In addition, the phorbolester PM_A (final concentration 1.0 ~tM) also induced increase in [3H]thymidine uptake in Ca glioma cells (Fig. 3). As further shown in Figure 3, no © Harcourt Brace and Co. Ltd 1998
CCK-8S-induced proliferative action
187
90 kDa
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Fig. 1 PKC subspecies 0~, ~1, ~ll, ~', 5, s and ( are present in rat glioma C 6 cells. C 6 cell lysate was separated by 10% SDS-PAGE and transferred to nitrocellulose strips. PKC subspecies were estimated as described in Materials and methods. Migration of molecular mass marker (90 kDa, Santa Cruz) is indicated on the left.
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Materials and methods. The membranes were analysed by immunoblotting with polyclonal antibodies against the respective subspecies of PKC (1 #g/ml). Migration of molecular mass marker (90 kDa) is indicated on the left. -, non-stimulated MIA-PaCA-2 cells; +, C 6 cells cells stimulated with CCK-8S (10 riM)
additional increase in [3H]thymidine incorporation was observed after simultaneous stimulation of C 6 glioma cells with PMA (final concentration 1.0 gM) and CCK-SS (final concentration 10 nM) compared with the effect of PMA alone. When C6 cells were exposed to CCK-8S (final concentration 10 nM) significantly enhanced cell numbers could be noted in comparison with non-treated C 6 cells (Fig. 4). This effect could be mimicked by the phorbolester PMA (final concentration 1.0 gM) and totally blocked when Ca cells were preincubated with the PKC inhibitor BIM (1.0 ~tM) for 20 rain (Fig. 4).
© Harcourt Brace and Co. Ltd 1998
DISCUSSION Earlier studies have described high affinity CCKB-type receptors and their connection with inositol metabolism, 7 calcium signalling6, 7 and protein phosphorylation7 CCK8S-induced increase in [3H]thymidine incorporation in rat glioma Ca was also reported. 8 However, the involvement of protein kinase C activation in CCK action on C6 cells has not been demonstrated to date. It is well known that receptors that use calcium as a second messenger hydrolyse phosphoinositides resulting Neuropeptides (1998) 32(2), 185-189
188 Kaufmann et al
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in generation of InsP 3 and diacylgycerol. The InsP 3 release in the cytoplasm mobilizes calcium from internal stores, whereas diacylglycerol activates protein ldnase C. ~5 Members of the PKC family play a key regulatory role in a variety of cellular functions, including cell growth and differentiation. 1¢17 The purpose of the present study was to investigate the involvement of PKC in CCK-8S-induced growth effects in rat glioma C a cells. When C a cells were exposed to CCK8S activation of b o t h the conventional PKC isoforms [~I, ~II, 7 requiring calcium and diacylglycerol, and the atypical isoform ~, being b o t h calcium and diacylglycerol independent could be observed. It is known that the mitogenic effect of growth factors and phorbolesters m a y be mediated through PKCY ,2° Because CCK-8S-induced increase in [3H]thymidine incorporation and cell n u m b e r could be blocked by the PKC inhibitor bisindolylmaleimide, the involvement of protein ldnase C in CCKmediated growth action in C a glioma cells is highly indicated. This was supported by the findings that the phorbolester PMA mimicked the CCK-8S-induced effect on C a cell growth and no additive effect of CCK-SS on PMA-induced [3H]thymidine incorporation could be observed in C a glioma. To summarize, the involvement of protein kinase C in cholecystokinin-induced proliferative action in rat glioma C a cells has been demonstrated. Further investigations in this field are in progress, including b o t h the role of the individual PKC subspecies and their connecNeuropeptides (1998) 32(2), 185-189
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Fig. 3 [3H]thymidine incorporation in rat glioma Cs cells exposed to different experimental reagents. Cells were loaded with [methyl3H]thymidine as described in Materials and methods and radioactivity was counted by liquid scintillation. Control: [~H]thymidine incorporation in non-stimulated Ce cells. Values are the means _+SE of three independent experiments, each done in triplicate.
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Fig. 4 Effect of CCK-8S, PMA and bisindolylmaleimide I on the proliferation of C6 glioma cells. Experiments were performed as described in Materials and methods. Values represent the mean + SE percentage increase in cell number of at least three independent experiments, each performed in duplicate. Control: non-stimulated C6 cells. Data are presented as percentage of control (cell number = 467 + 21).
tion with signalling events downstream of the protein kinase C, including Raf and the MAP kinase pathway. ACKNOWLEDGEMENT
This study was supported partly by Grant II B6-Ka 1098/1-2 from the Deutsche Forschungsgemeinschaft (DFG). REFERENCES
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