Effects of ethanol exposure on neuropeptide-stimulated calcium mobilization in N1E-115 neuroblastoma

Alcohol, Vol. 10, pp. 83-88, 1993

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Effects of Ethanol Exposure on Neuropeptide-Stimulated Calcium Mobilization in N1 E- 115 Neuroblastoma T H O M A S L. S M I T H

Research Service (151), Department o f Veterans Affairs Medical Center Tucson, Arizona, 85723 and the Department o f Pharmacology, College o f Medicine, University o f Arizona, Tucson, Arizona 85724 Received 21 M a y 1992; Accepted 28 A u g u s t 1992 SMITH, T. L. Effects of ethanol exposure on neuropeptide-stimulated calcium mobilization in NIE-I15 neuroblastoma. ALCOHOL 10(1) 83-88, 1993.--The effects of acute and chronic (100 mM for 7 days) ethanol exposures on resting intracellular free calcium, [Ca2+]i, as well as bradykinin and neurotensin mediated [Ca2+]i mobilization were determined in intact NIE-115 neuroblastoma. [Ca2+]~was monitored fluorometrically with the calcium indicator, fluo-3/AM. Acute exposure to ethanol resulted in an inhibition of bradykinln mediated [Ca2+]i mobilization with significant effects observed only at 400 mM ethanol. Neurotensin mediated [Ca2+]i mobilization was not significantly affected by any of the ethanol concentrations tested. Similarly, resting [Ca2+]i(64 =t: 2 nM) was unaffected by either chronic or acute ethanol as high as 400 raM. However, chronic exposure to ethanol significantly reduced the magnitude of bradykinln mediated [Ca2+]i mobilization both in the absence and presence of extracellular [Ca2+]. In contrast, [Ca2+]~mobilization in the presence of various concentrations of neurotensin was not significantly affected by chronic ethanol exposure. The results suggest that neuropeptide mediated [Ca2+]~ mobilization is relatively insensitive to the acute presence of ethanol. In addition, chronic ethanol exposure appears to have selective effects on receptor mediated [Ca2+]i mobilization because this response to bradykinin, but not neurotensin, was significantly reduced in cells exposed to ethanol. The results also suggest that the reduction in bradykinin stimulated [Ca2+]i mobilization in chronically exposed cells is due in part to an inhibition of the release of intracellularly bound [Ca2+]. Bradykinin

Neurotensin

Intracellular free calcium

Ethanol

BECAUSE calcium plays a central role in modulating neuronal function, the ability of ethanol to alter calcium disposition has been the focus of several investigations. Studies utilizing synaptosomes (4,7,22) and PCI2 pheochromocytoma cells (21) have demonstrated that acute ethanol exposure leads to an increase in the resting level of intracellular free calcium, [Ca2+]i, and in addition inhibits KCl stimulated calcium influx through voltage-sensitive calcium channels. However, relatively high ethanol concentrations (i.e., > 100 mM) were required to produce these effects. Chronic ethanol exposure, on the other hand, has no effect on synaptosomal [Ca2+]~ (5). The increase in resting synaptosomal [Ca2+]i in the presence of acute ethanol is thought to be the result of a direct effect on calcium release from the endoplasm reticulum rather than through a mechanism involving inositol trisphosphate (IP3) (6,14). In addition to its effects on voltage-sensitive calcium channels, acute ethanol exposure has also been shown to inhibit muscarinic cholinergic receptor-mediated calcium mobilization in PC12 cells, but again the concentrations of ethanol" required to produce this effect were 100 mM or greater (21,25). In contrast, recent studies have demonstrated that acute ethanol in the 10-50 mM range produces significant

N1E-115neuroblastoma

inhibition of N-methyl-D-aspartate (NMDA) receptor mediated 45Ca2+ uptake into cerebellar granule cells (11). Similar results were obtained in hippocampal cells (13), dissociated brain cells (8) and more recently, in isolated sensory neurons (30). With the exception of these two receptor types, the effects of ethanol on calcium mobilization by other receptor types have not been determined to date. Because this laboratory has recently reported that acute and chronic ethanol exposures inhibit bradykinin-mediated phospholipase C activity in N I E 115 cells (27), the goals of the present investigation were to determine whether neurotensin and bradykinin-mediated calcium mobilizations were affected in a parallel manner after acute and chronic ethanol exposures. In addition, the effects of extracellular Ca 2+ and phorbol ester pretreatment on the [Ca2+]i responses to both peptides were determined. METHOD

Tissue Culture Mouse neuroblastoma (NIE-115) cells were cultivated essentially as described previously (27). Briefly, cell passages 14-24 were grown in 75 crn2 tissue culture flasks in 20 ml 83

84

SMITH

Dulbecco's modified Eagle's medium (DMEM) supplemented with 10070 (v/v) fetal bovine serum (Hyclone, UT), 50 gg/ml streptomycin, and 50 units/ml penicillin (Sigma Chemical Co., St. Louis, MO) at 37°C in a humidified atmosphere of 93070 air - 7.2070 CO2. In the chronic ethanol experiments, 20 ml of fresh medium containing 100 mM ethanol was added daily to each flask for 7 days. Confluent cells from control and ethanol treated flasks were detached with modified Puck's D1 solution and harvested immediately before each experiment. Cell viability for both control and ethanol treated cells was assessed by trypan blue exclusion and averaged greater than 85070.

Intracellular Free Calcium, [Cag+]~, Determinations [Ca2+]~ was determined essentially as described previously (10). Harvested cells from one flask (8-12 × 106 cells) were washed twice with 5 ml of a 10 mM HEPES buffer containing: NaC1, 110 raM; KC1, 5.3 mM; MgC12, 1 mM; sucrose, 80 raM; glucose, 25 mM, pH 7.4 and adjusted to 340 mOsM (Buffer A). Washed cells (4-6 × 106) were resuspended in 1.25 ml DMEM and incubated for 30 min at 35°C with 9 #1 of a 1.25 mM stock solution of fluo-3/AM (Molecular Probes, Eugene, OR) dissolved in DMSO containing 3.6°70 (w/v) pluronic acid. All washings and resuspensions were performed at 35 °C. Fluorescence determinations were made using a T-format series 300 spectrofluorometer (H & L Instruments, Burlingame, CA) with excitation wavelength set at 506 nm. An emission wavelength of 526 nm was achieved with a narrow band pass filer (Microcoatings, Westford, MA). Fluorescence intensity, F, was monitored continuously with an online IBM compatible personal computer. Immediately prior to each determination, a 200-250 gl aliquot of the cell suspension was washed twice with 5 ml of Buffer A, resuspended in 3 ml of Buffer A containing 1.8 mM CaC12 and transferred to a quartz cuvette. After the cell suspension reached 35°C, indicated concentrations of drugs were added and the subsequent change in F recorded. Free intracellular calcium was calculated as described previously (29) using a KD of 450 nM for fluo-3/AM (16).

F

[Ca2+li = KD •

-

days (data not shown). Figure 1 illustrates the inhibitory effects of acute ethanol exposure of the magnitude of [Ca2+]~ mobilization by a maximally effective concentration (10 8M) of bradykinin and neurotensin. Although ethanol appears to inhibit bradykinin stimulated [Ca2+]~ in a concentrationdependent manner, Dunnett's test revealed that significant inhibition (p < 0.05) was achieved only in the presence of 400 mM ethanol. In contrast, neurotensin stimulated [Ca2+]~ was not significantly affected by any of the ethanol concentrations tested (Dunnett's test, ns).

Chronic Ethanol Effects In other experiments, the effects of chronic ethanol exposure on bradykinin-stimulated [Ca2+]i were determined. Concentration-response curves were generated for both control and ethanol treated cells as shown in Fig. 2. Two way analysis of variance (ANOVA) showed a significant effect of bradykinin concentrations in control and ethanol treated cells, F(3, 16) = 41.0, p < 0.01. Computer analysis of control and ethanol curves yielded ECs0 values for bradykinin of 1.7 _ 0.9 × 10-gM and 2.7 + 1.3 x 10-gM, respectively. These values did not differ significantly. However, chronic ethanol exposure had a highly significant inhibitory effect on the [Ca2+]i response curve to bradykinin as revealed by ANOVA, F(I, 16) = 32.3,p < 0.01. In experiments designed to determine whether the effect of chronic ethanol on [Ca2+]~ mobilization was selective for bradykinin [Ca2+]i responses to neurotensin were also evaluated (Fig. 3). Analysis of the control curve for neurotensin yielded an average ECs0 value of 1.3 × 10-gM, which is in accord with previously published values for neurotensin stimulated [Ca2+] i in NIE-115 cells (28) and in adenocarcinoma HT29 cells (2). Two-way ANOVA indicated that chronic ethanol exposure has no significant effect on the [Ca2+]~ response to neurotensin, F(1, 24) = 0.13, ns. Because chronic ethanol exposure had a selective effect on bradykinin mediated [Ca2+]~ mobilization, additional experi-

O-SM)

Fmi n

Fmax - F

Separate calibrations were performed on each sample. Fmin was determined in the presence of 0.1% SDS and excess EGTA. Fmax was then determined after the addition of a saturating concentration of calcium. Total fluorescence was corrected for both autofluorescence and dye leakage prior to the calculation of [Ca2+]i.

:s Neurotensin (10"8M)

I

RESULTS

Acute Ethanol Effects Resting (unstimulated) [Ca2÷]~in control N 1E- 115 cells averaged 64 +_ 2 nM. These values are in close agreement with resting [Ca2÷]i values reported previously with fura-2 in human neuroblastoma cells (12) and in NG 108-15 hybrid cells (18). Acute 4-5 min exposures to ethanol concentrations as high as 400 mM had no significant effect on resting [Ca2+]i. Similarly, resting [Ca2+]i was unaltered in N1E-I15 cells which were previously exposed to 100 mM ethanol for seven

I~

I 200

I 3~

I 400

Ethanol (raM)

FIG. 1. Inhibitory effects of acute ethanol on the magnitude of [Ca2+]i mobilization by bradykinin and neurotensin. Control ceils were exposed to the indicated concentrations of ethanol 4-5 min prior to the addition of a maximally effective concentration of bradykinin or neurotensin (1 x 10-8 M). ~ [Ca2+]irepresents the difference between resting [Ca2+] i and that calculated from the peak fluorescence intensity observed (10-15 s) after the addition of agonist. Data are expressed as the mean + SEM from at least four independent observations. *indicatesp < 0.05 (Dunnett's test).

ETHANOL AND CALCIUM MOBILIZATION

85 Neuretensln

Bradyklnln

7070

T

c~trots

60--

._

50--

~ <1

40

*

/

f~

se

30 20 ~ p
10 0 10

i

I

I

I

9

8

7

6

FIG. 2. Concentration-response curves for bradykinin stimulated [Ca2+]i mobilization in control cells (open circles) and cells chronically exposed to ethanol (closed circles). Confluent cells were incubated in the absence or presence of 100 mM ethanol added daily to the culture media for 7 days prior to the [Ca2+]i determinations. A [Ca2+]~ was defined as in Fig. 1. Values represent the mean ± SEM from at least four independent observations performed on different days. The calculated mean ECho value for control and ethanol-treated groups were 1.8 x 10-~vl and 2.7 x 10 -9 M, respectively. Two-way ANOVA indicated a highly significant effect of ethanol on the [Ca2+]i responses to bradykinin (/7 < 0.01). *indicates p < 0.05; **p < 0.01, (Student's test).

ments were performed to determine whether both bradykinin and neurotensin utilize similar mechanisms to mobilize [Ca2+]. First, experiments were performed either in the absence or presence o f calcium in the buffer. The results are shown in Fig. 4. In the absence o f extracellular [Ca2+], the bradykinin stimulated rise in [Ca2+]i was less than half o f that observed in the presence o f 1.8 m M extracellular Ca 2+ for both control and ethanol treated cells. Moreover, in the absence o f extracellular Ca 2÷, the magnitude o f the bradykinin-

,o 30

~

'°

T

// ,o

J~

11

9

÷

+

1.8 m M Extraceilular Ca 2.

-Log Bradykinin

~

÷

8

7

- Log Neurotensin

FIG. 3. Concentration response curves for neurotensin stimulated [Ca2.]i mobilization in control (open circles) and ethanol treated cells (closed circles). Ethanol treated cells were exposed to 100 mM ethanol for 7 days as in Fig. 2. Values represent the mean + SEM from at least four independent observations performed on different days. The calculated mean ECso value for control cells was 1.3 x 10 -9 M. ANOVA indicated no significant effect of ethanol on the [Ca2+]i response curve to neurotensin.

FIG. 4. Influence of extracellular calcium on the magnitude of [Ca2+]~ mobilization by bradykinin and neurotensin. Cells were washed twice and resuspended in a HEPES-buffered salt solution (Buffer A) either in the absence or presence of 1.8 mM Ca 2+ . Stimulation of [Ca2+]i was achieved with either 10 -8 M bradykinin (panel A) or 10 -8 M neurotensin (panel B) as in Fig. 1. Values represent the mean + SEM from four independent observations. *indicates p < 0.01 when compared to the correspondng control values for bradykinin; **/7 < 0.01 when compared to stimulation by neurotensin in the presence of 1.8 mM Ca 2+ (Student's t test).

stimulated rise in [Ca2+]i was significantly less in the ethanol treated cells when compared to control values obtained under the same incubation conditions (Fig. 4A). Similarly, the neurotensin stimulated rise in [Ca2+]i in the absence of extracellular Ca 2+ was approximately half o f that observed in the presence o f extracellular Ca 2+ (Fig. 4B). These results demonstrate that both bradykinin and neurotensin receptor activation stimulates Ca 2+ influx as well as intracellular Ca 2+ release. In addition, the results suggest that the inhibition o f bradykinin stimulated [Ca2+]i mobilization in chronically exposed cells is due, in part, to an inhibition of intracellular Ca 2+ release. The influence o f protein kinase C on [Ca2+]i mobilization by both bradykinin and neurotensin was determined in experiments in which control cells were subjected to a short (45 min) exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). The results are summarized in Table 1. The rises in [Ca2+]i in response to both bradykinin and neurotensin were significantly inhibited in cells pretreated with P M A . Moreover, this inhibition was observed whether or not extracellular Ca 2+ was present. The results observed in the absence o f extracellular Ca 2+ suggest that protein kinase C activation reduces the amount o f receptor-mediated [Ca2+]i release from internal stores. In addition, these results demonstrated that the [Ca2+] i responses to both peptides are under similar regulation by protein kinase C. DISCUSSION The direct effects o f acute and chronic ethanol exposures on Ca 2+ disposition were determined in N I E - 1 1 5 neuroblastoma, a cell line which provides a homogeneous population o f neuronal-like cells. The results demonstrate that in these cells, normal resting [Ca2+]i was not significantly altered by either acute ethanol exposure as high as 400 m M or chronic exposure to 100 m M ethanol for 7 days. Similarly, acute ethanol (100 mM) has been reported previously to have no effect on resting [Ca2+]i in freshly dissociated brain cells (8). Moreover, ethanol

86

SMITH TABLE 1 AICa2+]t (nM) Controls ExtracellularCa2+ 10-SM Bradykinin 10-SMNeurotensin

100 nM PMA

+

-

+

-

58 + 4 40 + 5

26 + 2 21 _+ 2

18 + 3* 23 +_ 2*

12 ± 3I 11 ± 21

Effects of phorbol 12-myristate 13-acetate (PMA) on bradykinin and neurotensin stimulated [Ca2+]~ in the absence or presence of 1.8 mM extracellular Caz+. Control N1E-I15 cells were exposed for 4-5 min with 100 nM PMA prior to stimulation with the indicated neuropeptides. A[Ca2+]~was determined as in Fig. 2. Values represent the mean + SEM from 3-9 individual experiments. *Indicates p < 0.01 when compared to corresponding control values in the presence of buffer Ca2÷; "pp < 0.01 when compared to control values in the absence of buffer Ca2÷. (Student's t test, unpaired.)

as high as 600 mM was also without effect on resting [Ca2+]i of isolated hepatocytes (4). These findings differ from those earlier reported for PC12 cells (21) in which acute exposure to 100 mM ethanol was sufficient to significantly elevate resting [Ca2+]i. Whether various cell lines of neuronal origin exhibit differential Ca 2+ sensitivities to ethanol will require future investigation. Our results also differ from those utilizing synaptosomal preparations. Although ethanol elicited a significant increase in synaptosomal [Ca2+]~, concentrations as high as 350-500 mM were required to demonstrate this effect (4,22). Because ethanol concentrations in this range are generally considered lethal, the relevancy of these effects to ethanol intoxication may be questionable. Moreover, conclusions drawn from synaptosomal data may be confounded in that intrasynaptosomal [Ca2+]i can be increased simply by raising the concentration of Ca 2+ in the bathing media (4,26), suggesting that the synaptosomal preparations are inherently "leaky" to calcium. Bradykinin induced [Ca2+] i mobilization observed in the present investigation was also relatively insensitive to acute exposure to ethanol in that an ethanol concentration of 400 mM was required to produce a significant inhibition of this response. Although the [Ca2+]i response to neurotensin tended to be inhibited by acute ethanol, the inhibition failed to achieve statistical significance even in the presence of 400 mM ethanol. These results suggest that receptor-mediated [Ca2+] i mobilization, presumably through phospholipase C activation, is minimally affected by physiologically relevant concentrations of ethanol. The inhibitory effect of chronic ethanol exposure on bradykinin-stimulated [Ca2+]i mobilization observed in the present study parallels previous observations from this laboratory (27) as well as others (24) in which neuroblastoma cells chronically exposed to ethanol exhibited a significant decrease in the formation of [3H]inositol phosphates elicited by bradykinin. Furthermore, in the absence of extracellular Ca 2+ , the bradykinin induced rise in [Ca2+] i was still significantly inhibited in cells chronically exposed to ethanol when compared to control cells stimulated under the same conditions. These results suggest that chronic ethanol affects the amount of bradykinin-induced release of intracellularly bound Ca 2+, presumably reflecting reduced InP3 production by bradykinin. Our results also demonstrate that the effect of chronic ethanol is selective, at least with respect to neurotensin, because neither the neurotensin

induced rise in [Ca2+]i nor the neurotensin induced increase in [3H]inositol phosphate production (27) was affected by chronic ethanol. Attempts were made in the present investigation to identify mechanisms which may underlie the selective inhibitory effects of chronic ethanol. Experiments performed in the absence or presence of extracellular Ca 2+ indicate that both bradykinin and neurotensin utilize Ca 2+ influx as well as intraccllular Ca 2+ release to elicit the observed rise in [Ca 2+]i. Both peptides stimulated intracellular Ca 2+ release to a similar extent. Although it was not possible to directly determine Ca 2+ influx per se for a given sample, Ca 2+ influx elicited by bradykinin, calculated as the difference between mean A [Ca2+]i values in the presence and absence of external Ca 2+, appeared greater than that elicited by neurotensin (see Table 1). Thus, it is possible that in N1E-115 cells, bradykinin receptors are coupled to other calcium channels in addition to those associated with the phospholipase C-InP3 pathway. Evidence for the coupling of bradykinin receptors to multiple Ca 2+ channels has been reported recently in PCI2 cells (9). The calculated mean values for Ca 2+ influx suggest that chronic ethanol exposure may also have reduced this component of the bradykininmediated rise in [Ca2+]i (see Fig. 4). Direct determination of Ca 2+ influx using 45Ca2+ will be necessary to confirm this possibility. Another possible locus for the selective effects of chronic ethanol exposure may be protein kinase C, because this enzyme has been reported to be altered in neural tissues chronically exposed to ethanol (1,15). Moreover, it is well established that the bradykinin-induced rise in [Ca2+]i is regulated by protein kinase C in PC12 cells (9), NG 108-15 neuroblastoma x glioma cells (20), and in NCB-20 neuroblastoma x Chinese hamster brain cell hybrid cells (19). Therefore, experiments were performed in the present investigation to determine whether the [Ca2+]i response to neurotensin was under similar regulation. Our results using P M A demonstrated that both bradykinin and neurotensin-stimulated [Ca2+]i mobilization are under similar inhibitory regulation by protein kinas¢ C, suggesting that this enzyme is an unlikely candidate for the differential effects of chronic ethanol on this component of the A [Ca2+]i response to bradykinin. An interesting observation here is that bradyldnin stimulated Ca 2+ influx appears to be more sensitive than [Ca2+]i mobilization to the effects of protein kinase C activation. Again, direct determination of

ETHANOL AND CALCIUM MOBILIZATION

87

Ca 2+ influx using 45Ca2+ is needed to establish the statistical significance of this observation. Although the exact locus of ethanol's actions on receptormediated release of intraceUularly bound Ca 2+ remain elusive, evidence suggests that chronic ethanol exposure affects sites distal to the bradykinin receptor because chronic ethanol has been reported to have no effect on [3H] bradykinin binding in NIE-115 cells (27) or in NG-108 neuroblastoma x glioma cells (23). In addition, the inositol 1,4,5-trisphosphate receptor is an unlikely candidate for the actions of chronic ethanol because an alteration in the binding parameters of this receptor should result in a generalized effect on Ca 2+ release induced by both neuropeptides. A more likely locus for the inhibitory effects of ethanol on bradykinin-mediated [Ca2+]i mobilization is the coupling of the bradykinin receptor to guanine-nucleotide binding protein (Gp) associated with phospholipase C. Recently, others have reported that the stimulation of phospholipase C activity by guanosine 5'-O(3-thiotriphosphate), GTP[S], in permeabilized NG108-15 cells is reduced after chronic ethanol exposure (23). Yet in NIE-115 ceils, the ability of GTP[S] to stimulate phospholipase C activity was unaffected by chronic ethanol (27). Furthermore, if the Gp of N1E-115 cells were affected by chronic ethanol exposure, one would predict a heterologous desensitization of phospholipase C mediated responses to both bradykinin and neurotensin in contrast to the selective effect

observed in the present investigation. It is important to note, however, that other investigators using neuroblastoma cells lines have shown that chronic ethanol exposure can affect the expression of the alpha subunit of both stimulatory (G-s) and inhibitory (Gi) GTP binding proteins which are coupled to adenylate cyclase. More important, these effects are selective for specific neural cell lines (3). Therefore, it is possible that the effects of chronic ethanol on the Gp binding protein per se are also cell-line specific. Further experiments utilizing other neuroblastoma cell lines is necessary to verify this hypothesis. In any case, recent results from this laboratory (27) as well as the present investigation, suggest that in NIE-115 cells, chronic ethanol exposure may alter the coupling of the bradykinin receptor to its recognition site on Gp, thereby reducing both bradykinin-mediated [3H]inositol trisphosphate production and subsequent InP3 dependent intracellular Ca z+ release without directly affecting the interaction of Gp with phospholipase C. Experiments designed to directly demonstrate the effects of chronic ethanol on the coupling of bradykinin and neurotensin to Gp are currently in process. ACKNOWLEDGEMENTS This project was supported by a medical research grant from the Department of Veterans Affairs. I thank Lucia Hoerr and Cindy McCollum for their expert technical assistance.

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SMITH 28. Snider, R. M.; Forray, C.; Pfenning, M.; Richelson, E. Neurotensin stimulates inositol phospholipid metabolism and calcium mobilization in murine neuroblastoma clone NIE-115. J. Neurochem. 47:1214-1218; 1986. 29. Tsien, R. Y.; Pozzan, T.; Rink, T. J. Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol. 94:325-334; 1982. 30. White, G.; Lovinger, D. M.; Weight, F. F. Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron. Brain Res. 507:332336; 1990.