Journal oj Neurochemisfry

Raven Press, Ltd., New York 0 199I International Society for Neurochemistry

Rapid Communication

Dantrolene Prevents Glutamate Cytotoxicity and Ca2+ Release from Intracellular Stores in Cultured Cerebral Cortical Neurons Aase Frandsen and Arne Schousboe PharmaBiotec Research Center, The Neurobiology Unit, Department of Biology B, The Royal Danish School of Pharmacy, Copenhagen, Denmark

Abstract: Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate-induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate-induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamatebinding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellularstores is essential for the propagation of glutamateinduced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest. Key Words: Cultured Neurons-Glutamate-Neurotoxicity-Neuroprotection-Calcium-Dantrolene. Frandsen A. and Schousboe A. Dantrolene prevents glutamate cytotoxicity and Caz+release from intracellular stores in cultured cerebral cortical neurons. J. Neurochem. 56, 1075-1078 (1991).

Dantrolene (1 -[ [5-(p-nitrophenyl)furfurylidene]amino]hydantoin, Na'), which in several cell types decreases or prevents mobilization of intracellular calcium stores (Kojima et al., 1984; Ward et al., 1986), has been shown to prevent this Ca2+ mobilization in cerebellar granule cells exposed to glutamate (Bouchelouche et al., 1989). Therefore, the possible protective effect of dantrolene against glutamate-induced neurotoxicity was tested in cultured neurons. Because cerebral cortical neurons are more susceptible to excitotoxicity than cerebellar granule cells (Frandsen and Schousboe, 1990) and glutamate is known to increase [Ca2'Ii in these neurons by an increased influx as well as by liberation of Ca" from intracellular stores (Wahl et al., 1989), it seemed advantageous to use such neurons instead of granule cells. To obtain information about the significance of the intracellular CaZ+stores in glutamate toxicity, the effect of dantrolene on this parameter was correlated with its effect on glutamate-mediated neuronal damage.

MATERIALS AND METHODS

The neurotoxicity of excitatory amino acids has during recent years been characterized qualitatively and quantitatively using primary cultures of different types of neurons as a model system (Frandsen and Schousboe, 1987, 1990; Choi, 1988; Frandsen et al., 1989). Hence, it has been shown that agonists for all the different glutamate receptor subtypes independently can induce cell lysis and death in neurons (Frandsen and Schousboe, 1987, 1990; Frandsen et al., 1989). The mechanisms by which these toxic actions are exerted are largely unknown, but an increased intracellular CaZ+concentration ([Ca2+Ii)is likely to play a crucial role (Rothman and Olney, 1986; Siesjo, 1990). In relation to this, it has recently been reported that mobilization of intracellular calcium stores may be of particular importance for glutamateinduced increases in [Ca2'Ii (Bouchelouche et al., 1989).

Materials Pregnant mice (15th gestational day) were obtained from the animal quarters at the Panum Institute, University of Copenhagen. Plastic tissue culture dishes were purchased from NUNC A/S (Roskilde, Denmark), and fetal calf serum was from Sera-Lab Ltd. (Sussex, U.K.). Cytosine arabinoside, poly-L-lysine, trypsin, trypsin inhibitor, DNAse, amino acids, vitamins, and dantrolene were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.), and enzymes and coenzymes were from Boehringer (Mannheim, F.R.G.). [3H]Glutamate (specific activity, 55 Ci/mmol) was obtained from Amersham International, PIC. (Buckinghamshire, U.K.). All other chemicals were of the purest grade available from regular commercial sources.

Received November 16, 1990; accepted November 19, 1990. Address correspondence and reprint requests to Dr. A. Frandsen at PharmaBiotec Research Center, The Neurobiology Unit, Department of Biology B, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2 I00 Copenhagen, Denmark.

Abbreviations used: [CaZ'li, intracellular CaZ+concentration;LDH, lactate dehydrogenase.

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A . FRANDSEN AND A . SCHOUSBOE

Cell cultures Cerebral cortical neurons were cultured essentially as described by Dichter (1978), Hertz et al. (1989), and Frandsen and Schousboe ( 1990).Astrocytic proliferation was curtailed by addition of the mitotic inhibitor cytosine arabinoside (20 pccM) after 48 h ofculture (Dichter, 1978; Larsson et al., 1985). The neurons were routinely cultured for 9 days before exposure to glutamate, KCI, and dantrolene. For further details regarding the characteristics of these cultured neurons. see Schousboe et al. (1985) and Drejer et al. (1987).

Determinations of cytotoxicity KCl (50 mM) or glutamate (10 prM) alone or in combination with dantrolene (30 pA4) was added to the respective culture media, and immediately before and at the end of the exposure period (9 h) aliquots were taken for determination of lactate dehydrogenase (LDH) activity. Sampling of aliquots and measurements of LDH activity were performed as previously described (Frandsen et al., 1989). Initially, dose-response relations of the protective effect of dantrolene as well as dantrolene toxicity per se were established over a concentration range from 1 to 250 p M . A dantrolene concentration of 30 pM was chosen because it was the lowest concentration at which protection against glutamate-induced toxicity was obtained. Dantrolene became toxic per se if the concentration exceeded 60 p M . In the media containing 50 m M KCI, an isosmotic amount of NaCl was omitted. To prevent a decrease in the extracellular concentration of glutamate during the exposure period, the nontoxic (Frandsen and Schousboe, 1990) glutamate uptake inhibitor (Drejer et al., 1982) L-aspartate-B-hydroxamate was added at 500 pM to the culture media during exposure to glutamate.

Measurements of [CaZfli All experiments were performed with neurons cultured for 9 days. Hue-3 acetoxy methyl ester loading and fluorescence measurements were performed according to the technique of Wahl et al. (1989). Calibration was performed with cells incubated with the ionophore A23187 (10 p M in HEPESbufferedsaline: 135 rnMNaCI, 5 mM KCI, 0.62 mM MgS04, 1.8 mMCaC12, 10 mMHEPES, and 6 mMglucose, pH 7.4). which allowed sufficient influx of Ca2+to attain the saturation level of binding with the intracellularly trapped fluo-3 ligand (FmaX). The fluorescence was quenched with 2 mhl CuC12 dissolved in 0.9% (wt/vol) NaCl plus 10 pMA23 187 to obtain the minimal fluorescence signal (F,," ). The observed relative fluorescence values for the cells were used in the following equation to calculate the free cytosolic Ca2+concentration: [Ca"], = KD ( F - Fm,")/(Fmax - F), where the KD is 450 nhf and F is the observed fluorescence,which increases on binding of Ca" without shifts in excitation or emission wavelengths (Minta et al., 1989). The neurons were exposed to the test compounds in HEPES-buffered saline. The minimal concentration of dantrolene needed to block intracellular release of Ca2+induced by glutamate ( 10 pA4) was 30 pM (tested over a concentration range from 1 to 250 p M ) .

Membrane preparation and [3H]glutamate binding assay Membrane preparations and binding assays using [3H]glutamatewere performed as described by Honor6 and Drejer (1988) and Wahl et al. (1 990), i.e., membranes were prepared from frozen cells by ultrasonic disintegration, centrifugations, and washes and used immediately for the binding experiments, which were performed in the following buffer: J Nrurochem , Chl 56, hro 3. 1991

30 mM Tris-HC1 and 2.5 mM CaC12, pH 7.1. Nonspecific binding was determined in the presence of 0.66 mM L-glutamate. Membranes were incubated with [3H]glutamate at 0°C for 30 min, and 5 ml of ice-cold buffer was added before filtration through Whatman GF/C glass fiber filters and washing twice, each with 5 ml of ice-cold buffer. Data analyses (McPherson, 1983) were performed using the software EBDA and LIGAND from Elsevier-Biosoft. B,, values are expressed on the basis of protein content in the membrane preparation as determined according to the procedure of Lowry et al. (I95 1).

RESULTS AND DISCUSSION Exposure of cerebral cortical neurons to glutamate in the presence of extracellular calcium resulted in a > 10-fold increase in [Ca2'], (Table I). In the simultaneous presence of dantrolene this increase could be reduced to 30%. If the cultures were exposed to glutamate in media without calcium added, the increase in [Ca2+Jiwas only 30% of that elicited by glutamate in the presence of extracellular Ca2+. Under these conditions dantrolene completely prevented the glutamate-induced increase in [Ca2+Ii(Table I). These results indicate that the glutamate-dependent increase in [Ca2+Iiis composed of at least two components, of which that directly related to influx of Ca2+may account for -70% of the total increase. Because the residual 30% is independent of extracellular Ca2+concentration, it is most likely originating from intracellularly released Ca2+,a process sensitive to dantrolene (Ward et al., 1986). Because dantrolene also prevented part of the increase in [Ca2+Iidependent on external Ca", it is possible that at least some of this Ca" also originates from a dantrolene-sensitive intracellular store, the release from which may be triggered by the glutamate-stimulated Ca2+ influx. Exposure of the neurons to 50 m M KCl led to an increase in [Ca2+Iisimilar to that seen after exposure to glutamate, but under these conditions dantrolene had no effect. This may indicate that intracellular Ca2+stores are not involved in the KCI-induced increase in [Ca2+Ii,a notion compatible with the observation that the KC1-induced increased in [CaZ+li was strictly dependent on extracellular CaZf(Table I). The finding that the effects of KCl and glutamate were additive (Table I)also supports the conclusion that both compounds increase [Ca2+Iibut via different mechanisms. The effect of dantrolene on glutamate-induced cytotoxicity was subsequently investigated. Dantrolene (30 p M ) , which per se did not exert any toxicity, completely prevented the toxic effects of glutamate (Table 2). Exposure of the neurons to 50 mMKCl did not result in leakage of LDH to the culture media. This is in agreement with the notion that depolarization per se is not of major importance for toxicity developing during prolonged exposure to glutamate (Rothman, 1984; Choi, 1985). Under conditions where a considerable component of the observed increase in [Ca2+Iiconsists of influx of external CaZ+, dantrolene completely protected against glutamate-induced cell damage. This indicates that the increase in [Ca2'Ii caused by influx and which does not involve intracellular stores is unlikely to be a determining factor in the induction of glutamate-related cytotoxicity. This is in accordance with the finding that exposure of the neurons to 50 mM KCI did not lead to cytotoxic damage in spite of the increase in [Ca2+Ii resulting from Ca2+influx. This conclusion is further in line with a recent report by Weiss et al. (1990) that the Ca2+L-

NE UROPROTECTION BY INHIBITION OF [Ca2+] RELEASE

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TABLE 1. Efects of 30 pM dantrolene on [Ca"], in cerebral cortical neurons exposed to glulamate, KCI, or both in the presence and absence of added extracellular Ca2+ [Caz+l, +CaCI, Addition

-~

Control

-CaCI2 Dantrolene

Control

Dantrolene ~

~

None Glutamate (10 p M ) KCI (50 mM) Glutamate (10 g M ) + KCl(50 mM)

75 rfr 3 1,070 rfr 13 700 f 11

1,676 f 9

71 k 3 380 k 7" 890 + 7 1,215 + 6

9 0 f 10 400 f 5 100 f 5 2

399

*

80 k 4 80 f 5" 89 f 7 78 k 7"

Experiments were performed with neurons cultured for 9 days. Following the loading procedure (see Materials and Methods), fluorescence was measured four times with 90-s intervals before test compounds were added in 50 p1 of HEPES-buffered saline with or without addition of CaC12.In the experimentswith 50 mMKC1an isomostic amount of NaCl was omitted. Data are average f SEM values from 12-24 experiments. Statistically significant difference from appropriate control values (Kruskal-Wallis test for nonparametric analyses; p < 0.00 I).

channel blocker (Sanguinetti and Kass, 1984; Godfraind et al., 1986) nifedipine could not prevent glutamate toxicity in similar cultures of neurons. O n the other hand, it can be concluded that mobilization of intracellular calcium stores must be centrally placed in the mechanisms responsible for the cytotoxic action of glutamate. To exclude the possibility that the effect of dantrolene could be secondary to interference with glutamate receptors, receptor binding studies were performed using membranes prepared from similar cultures of neurons. It was found that dantrolene did not affect the kinetic characteristics of [3H]glutamate binding because the KO values in the absence and presence of 30 p M dantrolene were, respectively, 205 23 and 199 f 30 nM. The corresponding B,,, values were 900 f 2 14 and 9 15 -t 89 fmol/mg of protein (mean -t SEM; n = 6). These values are in agreement with previously reported results (Wahl et al., 1991). Because glutamate is often associated with brain damage occumng after ischemic or epileptic episodes, the present finding that dantrolene acts as a neuroprotective agent may be of clinical relevance. In this context it is of importance

TABLE 2. Efects of 30 p M dantrolene on glutamateand KCI-induced leakage of LDH from cerebral cortical neurons LDH release (% of total)

Addition ~~

Control

Dantrolene

~~

None Glutamate (10 p M ) KCI (50 mM)

lot 1

11 f 2

75 f 2

15+ 1" 12f4

10

+3

All experimentswere performed with neurons cultured for 9 days. The cultures were exposed to KCI or glutamate alone or in combination with dantrolene(30 p M ) for 9 h by addition of the compounds to the culture media. To prevent a decrease in the extracellularconcentration of glutamate, the nontoxic (Frandsen and Schousboe, 1990) glutamate uptake inhibitor (Drejer et al., 1982) L-aspartate-0-hydroxamate was included at 500 p M . Sampling of aliquots and measurements of LDH activity were performed as previously described (Frandsen and Schousboe, 1987; Frandsen et al., 1989). For further details, see Materials and Methods. Data are average + SEM values from nine experiments. Statistically significant difference from the control value (KruskalWallis test for nonparametric analyses: p < 0.001).

that dantrolene is used clinically to prevent, e.g., malignant hyperthennia (for review, see Ward et al., 1986). Even though glutamate is likely to be the major mediator of excitatory amino acid-induced cell damage in vivo, the potential effects of dantrolene on toxicity and changes in calcium homeostasis induced by other excitatory amino acids in the neurons should also be investigated. Such studies may shed more light on the mechanisms involved and thus possibly facilitate the development of adequate treatments for the neurodegenerative disorders in which excitatory amino acids are thought to be involved.

Acknowledgment: Miss Charlotte F r e d s ~ eAndersen is cordially thanked for expert technical assistance. This work was supported by The Danish Biotechnology Program (19871990) and the NOVO, Lundbeck and Dir. Ib Henriksen Foundations.

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