Ethanol effects on bradykinin-stimulated phosphoinositide hydrolysis in NG 108-15 neuroblastoma-glioma cells

Alcohol, Vol. 6, pp. 475-479. ~ Pergamon Press plc, 1989. Printed in the U.S.A.

074t-8329/89 $3.00 + .00

Ethanol Effects on Bradykinin-Stimulated Phosphoinositide Hydrolysis in NG 108-15 Neuroblastoma-Glioma Cells P E R S I M O N S S O N , 1 G R A C E Y. S U N , * L A S Z L O V E C S E I AND CHRISTER ALLING

Department of Psychiatry and Neurochemistry, Lund University, Lund, Sweden and *Sinclair Comparative Research Farm, University of Missouri, Columbia, MO R e c e i v e d 6 M a r c h 1989; A c c e p t e d 21 June 1989

SIMONSSON, P., G. Y. SUN, L. VECSEI AND C. ALLING. Ethanol effects on bradykinin-stimulatedphosphoinositide hydrolysis in NG 108-15 neuroblastoma-glioma cells. ALCOHOL 6(6) 475-479, 1989. --The effect of short- and long-term ethanol exposure on bradykinin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIPe) was investigated in neuroblastoma × glioma hybrid cells (NG 108-15). Acute exposure of 50-150 mM ethanol neither influenced the bradykinin-stimulated accumulation of [3H]-inositol phosphates (IP 1, IP2, IP3) nor the hydrolysis of PIP2 in cells labelled with [3H]-inositol. Furthermore, ethanol (100 mM) added in the absence of agonist did not influence these parameters. However, in cells cultivated for 4 days in 100 mM ethanol, PIP: hydrolysis and IP~, IP2 and IP3 formation after stimulation by 10-6-10- ~ M bradykinin was markedly inhibited while there was no effect on the basal levels or on the levels found after stimulation with low concentrations of bradykinin. The inhibitory effect of ethanol on IP accumulation became significant after 2-3 days of ethanol. Ethanol Bradykinin NG 108-15 cell-line

Phosphatidylinositol 4,5-bisphosphate

ALTHOUGH several reports on the effects of ethanol on receptorstimulated phospholipase C activation has been published during the last two years, it has not yet been possible to elucidate a general mode of action of ethanol on this signal transduction pathway. A reduced formation of inositol phosphates after receptor-stimulation with different agonists have been found in [3H]inositol labelled brain slices both after acute and long-term exposition (7, 12, 13) although negative results have also been reported (27). On the other hand, ethanol was shown to cause a transient phospholipase C activation in hepatocytes and platelets (11,23). Recently, we have reported a potentiating effect of acute ethanol exposition on the serotonin-stimulated inositol lipid metabolism in primary rat astroglial cell-cultures (25). A similar effect with respect to ethanol exposure was also found when using noradrenaline to stimulate astrocytes (20). It thus seems likely that the ethanol effect is dependent not only on the type of receptor being studied but also on the tissue or cell being investigated. The selective receptor sensitivity to ethanol may be important in understanding ethanol effects on receptors in isolated brain regions (7,12). Literature on the long-term effects of ethanol also show variable results. Both inhibition (24), stimulation (12, 13, 20) and no effect at all (8,27) on receptor-stimulated [3H]-IPI formation have been reported.

Inositol phosphates

Phospholipase C

From the studies using brain-slice techniques, it is difficult to establish the cellular vulnerability to ethanol. Therefore, culture of primary cells or established cell-lines may be a better tool for elucidation of the complex mechanisms by which ethanol may exert its effects (21). The aim of this study is to investigate acute and chronic effects of ethanol on neuroblastoma-glioma hybrid cells (NG 108-15), a defined neuronal cell-line which is known to possess bradykinin receptors coupled to the hydrolysis of phosphoinositides (9, 10, 14, 26, 29). METHOD

Procedures for Cell Cultivation and Stimulation of [~H]-labelled NG 108-15 Cells With Bradykinin NG 108-15 cells (passage 20-33) were cultivated in 50 mm diameter plastic dishes (NUNC A/S, Denmark) containing 4 ml Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 2 mM 1-glutamate, 2% HAT 50X (final concentration: hypoxantine 0.1 raM, aminopterine 4 txM, thymidine 16 txM), 100 txg/ml streptomycin and 100 IE/ml penicillin. Medium, fetal calf serum and suppplements were from Flow Laboratories, U.K. while antibiotics were purchased from ASTRA Pharmaceuticals,

IRequests for reprints should be addressed to Per Simonsson, M.D., Department of Psychiatry and Neurochemistry, Lund University, St Lars Hospital, P.O. Box 638, S-220 06 Lund, Sweden.

475

476

SIMONSSON, SUN, VECSEI AND ALLtNG

Sweden. The culture dishes were maintained at 37°C in an incubator under humidified atmosphere containing t0% CO z and 90% air. Medium was changed daily. In experiments studying the effects of long-term exposure of ethanol, 100 mM ethanol was added directly to medium daily. The alcohol concentration in medium was reduced to approximately 50% of initial concentration after 24 hr of incubation. Cells to be labelled with [3H]-inositol were grown for 24 hr in 4 ml medium containing 1 IzCi myo-[3H]-inositol/ml (New England Nuclear, Boston, MA specific activity 15-20 Ci/mmot). Antibiotics were omitted during labelling and stimulation. Cells were washed 1 hr before stimulation and new medium containing 25 mM LiCI and, when indicated, ethanol was added. A 1 hr preincubation period was used as cellular morphology was slightly modified immediately after the change of medium. Normal morphology was obtained within 1 hr. Cells were then stimulated by adding bradykinin (Sigma Chemical Co, St. Louis, MO) at appropriate concentrations while maintaining the medium at 37°C using a hot plate calibrated to keep the cell cultures at a constant temperature. With the exception of the time-response experiments, all dishes were stimulated for 30 sec as this time has been shown to correspond to a significant hydrolysis of PIP 2 and accumulation of [3H]-inositol phosphates (26). After repeated washings with cold medium, cells were scraped in 0.5 ml Tris buffer with 1 mM EDTA and extracted with 2ml chloroform:methanol:HC1 (100: 200:2). Following the addition of 0.5 ml chloroform and 0.5 ml water, the lipid- and water-phases were separated.

Analysis of [3H]-Inositol Phosphates and [SHl-lnositol Lipids Inositol phosphates were separated essentially according to Berridge et al. (1) as described earlier (25,26): The diluted water-phase was applied to a column containing 0.5 g Dowex 1-8X in the formate form and free [3H]-inositol was eluted by adding 12 ml 10 mM inositol and 12 ml 5 mM disodiumtetraborate/60 mM sodiumformate. IPj, IP 2 and IP 3 were then eluted by adding increasing concentrations of ammonium formate in 100 rnM formic acid. An aliquot of each fraction was taken for scintillation counting. Inositol lipids were separated by evaporating the lipid-phase under N 2 and dissolving it in 1 ml chloroform:methanol (2:1). Aliquots were taken for the determination of total incorporation of [3H]-inositol into the inositol lipids. The lipids were then separated on oxalate-treated Silica Gel 60 HPTLC plates (Merck, Darmstadt, FGR) with a solvent system consisting of chloroform: methanot:conc. NH3:H20 (65:47:2.5:151. Lipids were visualized by exposure to 12 and the radioactivity of the scraped spots measured by liquid scintillation counting.

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RESULTS

Effect of Acute Ethanol Exposure To elucidate if acute ethanol exposure per se stimulates

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time (rain) FIG. t. Ethanol (100 raM) does not influence the basal levels of [:~H]-IP~ (O), [3H]-IP2 (F'l), [3H]-IP3 (O), [3H]-PIP (L~) or [3H]=PIPz (A) in NG 108-15 cells. Results are expressed as percent of total counts in the lipid-phase and are mean + SEM of 6 dishes at each time-point from two experiments.

phospholipase C, NG 108-15 cells were chaUanged with 100 mM ethanol for 30 sec to 5 min in the absence of agonist, It was found that this treatment did not influence the basal levels of in0sitol phosphates, PIP or PIP 2 (Fig. 1). Likewise, 150 mM ethanol for 15 min did not increase the hydrolysis Of PIP 2 or the formation of inositol phosphates (Table 1). Furthermore, ethanol at physiologically relevant concentrations (50-150 mM) neither influenced the bradykinin-stimulated formation of [3H]-inositol phosphates nor

TABLE 1 THE EFFECTOF ACUTEETHANOLEXPOSUREON BRADYKININ STIMULATEDINOSITOLLIPID METABOLISMIN NG 108-15 CELLS IP I BK (M) 0

Ethanol (mM)

IP~

IP~

PIPz

PIP

PI

(% Radioactivity of Total Counts in Lipids)

0

0.818 ¢0.06)

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0

1.704 0.244 (0.141 ~0.045

0.200 1.14 0.87 19.3 10.031 (0.111 10.131 (0.46)

10 ~

50

1.840 0.238 (0.14~ f0.061

0.176 (0.02,

1.04 0.82 19.0 (0.115 (0.125 (t.171

10 ~

100

1.916 0.296 *0.115 (0.06)

0.204 (0.04/

1.16 0.82 ¢0.121 (0.09)

20.6 (1.311

10 -6

150

1.830 0.272 (0.18/ (0.075

0.206 1.12 13.79 t0.031 (0.121 (0,09)

21.l (1.711

150

0.760 (0.05~

0. 118 1.54 ¢0.03) (0.07)

20.0 ~1.5)

Statistical Analysis To compensate for slight variations in cell number and incorporation of [3H]-inositol into inositol lipids, results were divided by the radioactivity in the total lipid pool from each culture dish and expressed as percent of total [3H]-inositol incorporation. No difference was found between the incorporation of [31-1]inositol into inositol lipids or inositol phosphates when comparing ethanol-treated and ethanol-naive cell cultures (Table 2). The bradykinin effect was calculated as the percent change compared to basal, unstimulated values. Statistical analysis was performed using Student's unpaired t-test.

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0

0.0870 (0.015

0.092 (0.011

0.13l 1.40 (0.01~ (0.14)

1.04 (0.07)

1.09 (0,095

20.6 (0,08)

[-~H]-inositol labelled cells were incubated tbr 15 min in 50, 100 and 150 mM ethanol and stimulated for 30 sec by 10 -6 M bradykinin. Data is expressed as % radioactivity of total labelling of the lipid.phase (total counts). Results are mean ~+__SEM) of 4 experiments, each done in triplicate.

ETHANOL AND INOSITOL LIPIDS

477

TABLE 2 THE EFFECT OF CHRONIC ETHANOL EXPOSURE OF NG 108-15 CELLS ON THE INCORPORATION OF [3H]-INOSITOL INTO THE PHOSPHOINOSITIDES AND INOSITOL PHOSPHATES

Control Dishes (cpm/dish) [3H]-PI [3H]-PIP [3H]-PIP2 [3H]-IPI [3H]-IP2 [3H]-IP3

42408 (2673) 1574 (116) 2201 (241) 2070 (354) 247 (19) 216 (18)

100.

Ethanol Dishes (cpm/dish) 37358 1651 2040 2299 253 219

(5483) (85) (288) (277) (16) (23)

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Cells were labelled for 24 hr with l /~Ci [3H]-inositol/ml medium. Ethanol-treated dishes were grown in 100 mM ethanol during the labelling and had been exposed to ethanol for three days prior to labelling. Results are expressed as cpm/dish and are mean ( -+SEM) of 4 experiments.

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the levels of [3H]-phosphoinositides (Table 1).

Effect of Long-Term Ethanol Exposure To study the effect of long-term ethanol exposure, NG 108-15 cells were grown for 4 days in medium containing 100 mM ethanol. The protein content was unaffected by 4 days of exposure (1.43_+0.08 mg/control dish vs. 1.51_+0.05 mg/ethanol dish, mean-+ SEM of 16 dishes). No significant effect was seen on the basal [3H]-labelling of inositol lipids or inositol phosphates (Table 2). After 4 days of ethanol exposure, the maximal rate of bradykinin stimulated formation of IP~, IP 2 and IP 3 was significantly reduced (Fig. 2). A significant inhibition of IP 3 formation was also found after stiulation with 10 7 M bradykinin, a concentration close to the estimated ECso. The hydrolysis of PIP, was also inhibited at this concentration (Fig. 3) while the effect on the metabolism of PIP and PI was obvious only at higher concentrations of bradykinin (Fig. 3). The inhibition of IP 2 and IP 3 accumulation became detectable after 2-3 days of ethanol exposure, while no significant inhibition of IP~ was found in this set of experiments (Fig. 4).

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DISCUSSION

Our results indicate that acute ethanol exposure at physiologically relevant concentrations does not influence basal or bradykinin-stimulated inositol lipid signal transduction in NG 108-15. This is in contrast to several reports on the inhibitory effect of ethanol on neurotransmitter-stimulated PIP 2 hydrolysis in brain slice preparations (7, 12, 13). Contrary to the results on bradykinin-stimulated NG 108-15 cells, we have recently found that the serotonin-stimulated response in primary rat astrocytes is potentiated by the same concentrations of ethanol as those used in this study (25). Similar results were also obtained by Ritchie et al. using noradrenaline-stimulated primary astrocytes (20). Since ethanol alone has been shown to stimulate phospholipase C in hepatocytes (11) and in platelets (23), these results speak against the hypothesis that ethanol acts on phosphoinositide metabolism via one single mechanism. The hypothesis that different receptors and/or cell types are differentially sensitive to ethanol is strengthened by our results from chronically exposed NG 108-15 cells. From the reports so far published on the chronic effects of ethanol, it is not possible to propose a general theory for the adaptive response of the phos-

bradykinin (-log M) FIG. 2. Ethanol exposure (100 mM) for 4 days inhibits the bradykininstimulated formation of [3H]-IP3 (a), [3H]-IP2 (b) and [3H]-IP l (c) in NG 108-15 cells. Cells were incubated for 1 hr with 25 mM LiCI before stimulation for 30 sec with 10-8-10 -5 M bradykinin. Solid lines indicate control dishes and broken lines are ethanol-exposed dishes. Results are expressed as percent increase above values from unstimulated dishes and are mean _+SEM of 12 dishes from 4 experiments. *p<0.05, **p<0.005, ***p<0.001 (Student's unpaired t-test). pholipase C signalling system (7, 8, 12, 13, 20, 27, 28). Although many receptor complexes are known to be linked to the polyphosphoinositide pathway, there is evidence of a difference in the way receptors respond to toxins, aluminium fluoride and phorbol ester treatment (2, 4-6, 15-17, 19). It is therefore likely that there may be a difference in the way individual receptors function in the presence of ethanol. The GTP-binding protein is one candidate

478

SIMONSSON, SUN; VECSEI AND ALLING

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bradykinin ( - l o g M) FIG. 3. Ethanol exposure (100 raM) for 4 days inhibits the bradykininstimulated metabolism of [3H]-PIP2 (a), [3HI-PIP (b) and [3H]-PI (c) in NG 108-t5 cells. Cells were incubated for 1 hr with 25 mM LiC1 before stimulation for 30 sec with 1 0 - s - t 0 - s M bradykinin. Solid lines indicate control dishes and broken lines are ethanol-exposed dishes. Results are expressed as percent change compared to values from unstimulated dishes and are mean±SEM of 12 dishes from 4 experiments. *p<0.05, **p<0.005, ***p<0.001 (Student's unpaired t-test).

which has been suggested as a target of ethanol effects. From studies on the effect of chronic ethanol on the adenylate cyclase second messenger system it has been demonstrated that the attenuated receptor-response is mediated via reduced formation of mRNA for the alpha-subunit of G~-protein (18). The G-proteins associated with phospholipase C has not yet been as well characterised but studies on platelets (23) but not hepatocytes (22), have shown that this G-protein can be stimulated by ethanol. Our observation that chronic ethanol reduced the maximal response to bradykinin can also be interpreted as an impairment of the Gprotein function. The discrepancies found between the effects of ethanol on NG 108-15 and serotonin- and noradrenaline-stimulated astrocytes may be explained by the fact that NG 108-15 cells are transformed hybrid cells and that they may lack essential components that are targets for the ethanol effects. This explanation can not be tested in this study but the bradykinin transduction system on NG 108-15 cells shares a similar pharmacological profile with the bradykinin system on primary astrocytes (3). Therefore, it is more likely to

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FIG. 4. The time-course of the inhibiting effect of 100 mM ethanol on bradykinin-stimulated formation of [3H]-tP3 (a), [3H]-IP2 (b) and [aH]-IP~ (c) in NG 108-15 cells. Cells were stimulated for 30 sec with 10 .-6 M bradykinin in the presence of 25 m2¢I LiCt. Solid lines indicate control dishes and broken cells exposed to 100 mM ethanol for 4 days. Results are expressed as percent change compared to values from unstimulated dishes and are mean---SEM of 9-12 dishes from 3-4 experiments. *p<0.05. **p<0.01, ***p<0:005 (Student's unpaired t-test).

suppose that the lack of an acute effect of ethanol is a characteristic of the bradykinin receptor complex. Data from e x p e ~ e n t s on primary astrocytes also indicate that acute ethanol, in contrast to its effect on the serotonin response, does not potentiate bradykinin-stimulated [3H]-IP formation (25). In conclusion, long-term ethanol exposure i ~ b i t s the phosphoinositide-derived transduction system coupled to the bradykinin receptor on a cell-line of neuronal origin. ACKNOWLEDGEMENTS We wish to thank Monica Mihailescu for her professional technical assistance and Dr. Pam Fredman, Department of Psychiatry and Neurochemistry, University of G0teborg, GOteborg, Sweden, for the gift of NG 108-15 cells. This project was suportedby: Swedish Medicat Research Council (Project No. 05249), The Bank of Sweden T e r e e n ~ Foundation, The A. P~lhlsson Foundation and the Medieat Faculty, Lund Uhiversity.

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E T H A N O L A N D INOSITOL LIPIDS

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