Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons

Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons

European Journal of Pharmacology, 241 (1993) 1-6 1 Elsevier Science Publishers B.V. EJP 53246 Protective effects of a vitamin B12 analog, methylco...

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European Journal of Pharmacology, 241 (1993) 1-6

1

Elsevier Science Publishers B.V.

EJP 53246

Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons Akinori Akaike, Yutaka Tamura, Yuko Sato and Takeharu Yokota Department of Neuropharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama UniL'ersity, Fukuyama 729-02, Japan Received 11 February 1993, revised MS received 2 June 1993, accepted 15 June 1993

The effects of methylcobalamin, a vitamin B12 analog, on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Cell viability was markedly reduced by a brief exposure to glutamate followed by incubation with glutamate-free medium for 1 h. Glutamate cytotoxicity was prevented when the cultures were maintained in methylcobalamin-containing medium. Glutamate cytotoxicity was also prevented by chronic exposure to S-adenosylmethionine, which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and S-adenosylmethionine also inhibited the cytotoxicity induced by N-methyl-D-aspartate or sodium nitroprusside that releases nitric oxide. In cultures maintained in a standard medium, glutamate cytotoxicity was not affected by adding methylcobalamin to the glutamate-containing medium. In contrast, acute exposure to MK-801, a NMDA receptor antagonist, prevented glutamate cytotoxicity. These results indicate that chronic exposure to methylcobalamin protects cortical neurons against NMDA receptor-mediated glutamate cytotoxicity. Cerebral cortex; Glutamate; Nitric oxide (NO); NMDA (N-methyl-D-aspartate); Methylcobalamin; Vitamin B j2

I. Introduction

Methylcobalamin is an active coenzyme of the vitamin B]2 analogs, that are essential for cell growth and replication (Hillman, 1990). Methylcobalamin is required for the formation of methionine from homocysteine. Methionine is transformed to S-adenosylmethionine in the methionine metabolism pathway. S-Adenosylmethionine plays an important role in the maintenance of a number of methylation reactions in both the peripheral organs and the central nervous system (Hall, 1990). Although the mechanisms responsible for the neurological lesions of vitamin B12 deficiencies are less well understooJ, vitamin Bl2 analogs, including methylcobalamin, have been widely used in therapy of neurological diseases not only in the peripheral nervous system but also in the central nervous system (Chanarin et al., 1985; Metz and Van der Westhuyzen, 1987). Recent evidence has suggested that certain excitatory amino acids (EAAs) such as glutamate play impor-

tant roles in the neurotoxicity observed in hypoxicischemic brain injury (Choi, 1988; Meldrum and Garthwaite, 1990). Moreover, brief glutamate exposure induces delayed death in cultured neurons of the cerebral cortex. It has been postulated that the N-methylD-aspartate ( N M D A ) receptor is the predominant route of glutamate neurotoxicity since selective antagonists of N M D A receptors possess protective effects against neuronal degeneration both in animal models of ischemia and in cultured neurons (Choi, 1988; Hartley and Choi, 1989). The objective of this study was to investigate the effects of methylcobalamin on neuronal damage in the central nervous system. We assessed the effects of methylcobalamin on glutamate-induced cell death using cultured neurons obtained from the cerebral cortex.

2. Materials and methods 2.1. Cell culture

Correspondence to: A. Akaike, Department of Neuropharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-02, Japan. Tel. 81-849-36-2111, ext. 5233, fax 81-849-36-2024.

Primary cultures obtained from the cerebral cortex of fetal rats were maintained and glutamate cytotoxicity was assessed as described previously (Akaike et al., 1991; T a m u r a et al., 1992). Briefly, single cells dissoci-

ated from the whole cerebral cortex of fetal rats (16-18 days gestation) were plated on plastic coverslips which were placed in Falcon 60-mm dishes. Cultures were incubated in Eagle's minimal essential salt medium (Eagle's MEM) supplemented with 10% heat-inactivated fetal bovine serum (1-9 days after plating) or 10% heat-inactivated horse serum (10-12 days after plating), glutamine (2 raM), glucose (total 11 mM), N a H C O 3 (24 mM), and H E P E S (10 mM). The cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere. After 8 days of plating, non-neuronal cells were removed by adding 10 -5 M cytosine arabinoside. Only mature (10-14 days in vitro) cultures were studied.

2.2. Measurement of neurotoxicity Neurotoxicity induced by EAAs was quantified by examining cultures under Hoffman modulation microscopy according to the methods described in our previous reports (Tamura et al., 1992, 1993). All experiments were performed in Eagle's MEM at 37°C. Cell viability was assessed using trypan blue exclusion. Ceils stained with trypan blue were regarded as non-viable. Over 200 cells were counted to determine the viability of a cell culture. The viability of the cultures was expressed as percentage calculated from of the ratio of the number of unstained ceils (viable cells) to the total number of cells counted (viable ceils plus non-viable cells). In each experiment, 5-10 coverslips were used to obtain means _+ S.E.M. of cell viability.

2.3. Evaluation of drug-induced protection against EAA cytotoxicity Protective effects of the drugs were assessed by chronic and acute drug application. To study chronic drug application, a drug was added to the incubation medium immediately after cell plating until immediately before EAA exposure. Drug-containing medium was changed every 48 h. In the preliminary experiments, we found no significant decrease in the methylcobalamin concentration in the medium within 48 h. The drug was removed from culture immediately before EAA treatment. Cell viability was occasionally affected by the chronic administration of the drug itself. To normalize the variation in the viability of non-treated and drug-treated cultures, protection induced by the chronic drug-administration was calculated using the following equation: protection ( % ) = (((A - C)-(B - D ) ) / ( A - C)) x 100, in which A is the viability of non-treated cultures (control), B is the viability of cultures treated with a drug alone, C is the viability of EAA-treated cultures and D is the viability of the cultures treated with a drug and EAA. An example of the calculation of the protection rate for

100 9( 8(

7c

(B- D)

:~ 5C

'-

C)

t~ 4C 3C 2C IC 0

AB Control

CD Glutamate

Fig. 1. An example of the protective effect of chronically applied methylcobalamin against glutamate cytotoxicity. (A) Viability of control cultures. (B) Viability of cultures chronically exposed to methylcobalamin (10 6 M): (C) Viability of cultures after a brief exposure to glutamate (1 mM). (C) Viability of methylcobalamin-treated cultures after a brief exposure to glutamate (1 mM). The value (A-C) represents the glutamate-induced decrease in the viability of cultures maintained in the standard medium. The value (B-D) represents the glutamate-induced decrease in the viability of cultures maintained in the methylcobalamin-containing medium.

chronic methylcobalamin administration is shown in fig. 1. To assess the effects of acute drug application, a drug was added to the medium containing EAA. This was replaced with EAA-free medium for 1 h. Protection by the acute drug administration was calculated using the following equation: protection ( % ) = ( ( D C ) / ( A - C)) × 100, in which A, C and D are same as in the former equation.

2.4. D ~ The following drugs (sources) were used. MK-801 (Research Biochemicals), monosodium L-glutamate (Nacalai tesque), methylcobalamin (synthesized at Eizai), N M D A (Sigma), S-adenosylmethionine (synthesized at Eizai), and sodium nitroprusside (Wako). The drugs were dissolved in Eagle's solution immediately before the experiments.

2.5. Statistics The data were expressed as means_+ S.E.M. The statistical significance of the data was determined by Dunnett's two-tailed test.

3. Results

3.1. Neurotoxicity induced by EAAs and sodium nitroprusside In previous experiments (Akaike et al., 1991; Tamura et al., 1992), we induced marked reduction of cell

TABLE 1 Cytotoxic effects of glutamate, NMDA and sodium nitroprusside on cultured cortical neurons. n is the number of coverslips. Abbreviations: Glu, glutamate; SNP, sodium nitroprusside. Treatment

n

Viability (%)

% of control

Control Glu 1 mM NMDA 1 mM SNP 0.5 mM

10 10 10 10

90.4 _+0.5 33.1 _+1.7 a 39.0_+0.9~ 35.4-+ 1.4 a

100 36.6_+ 1.9 43.1 _+1.5 39.2 _+1.5

P < 0.01 significantly different from control.

viability by exposing cultures to g l u t a m a t e (0.5-1 m M ) for 10 m i n followed by i n c u b a t i o n in g l u t a m a t e - f r e e m e d i u m for m o r e t h a n 1 h. T h e r e f o r e , in the p r e s e n t study, cultures were exposed as follows: 10 m i n to E A A or s o d i u m n i t r o p r u s s i d e followed by 1-h i n c u b a tion in E A A - f r e e or s o d i u m n i t r o p r u s s i d e - f r e e m e d i u m . T a b l e 1 s u m m a r i z e s the cytotoxic effects of glutam a t e (1 mM), N M D A (1 m M ) a n d s o d i u m n i t r o p r u s side (0.5 mM). G l u t a m a t e a n d s o d i u m n i t r o p r u s s i d e were a d d e d to the m e d i u m c o n t a i n i n g a n o r m a l conc e n t r a t i o n of Mg 2+ (1.6 mM). N M D A was a d d e d to the Mg2+-free m e d i u m since o u r previous study had d e m o n s t r a t e d that N M D A i n d u c e s cytotoxicity in the a b s e n c e of Mg 2+ ( T a m u r a et al., 1992). W h e n the cultures were m a i n t a i n e d with s t a n d a r d m e d i u m , brief exposure of the cells to either g l u t a m a t e or N M D A significantly r e d u c e d cell viability. S o d i u m n i t r o p r u s side, which s p o n t a n e o u s l y releases nitric oxide, also r e d u c e d cell viability to a m a g n i t u d e similar to that of EAAS.

3.2. Effects of chronic administration of methylcobalamin and S-adenosylmethionine T a b l e 2 s u m m a r i z e s the viability c h a n g e s in cortical cultures following c h r o n i c a d m i n i s t r a t i o n of methylc o b a l a m i n or S - a d e n o s y l m e t h i o n i n e . As described in Methods, cultures were exposed to the d r u g - c o n t a i n i n g m e d i u m i m m e d i a t e l y after p l a t i n g o n c u l t u r e dishes, a n d were t h e n m a i n t a i n e d in the d r u g - c o n t a i n i n g m e d i u m for 1 0 - 1 2 days. N e i t h e r m e t h y l c o b a l a m i n n o r S - a d e n o s y l m e t h i o n i n e at c o n c e n t r a t i o n s of 1 0 - ~ - 1 0 -5 TABLE 2 Effects of chronic administration of methylcobalamin and S-adenosylmethionine on the viability of cultured cortical neurons. n is the number of coverslips. Abbreviations: MB12, methylcobalamin; SAM, S-adenosylmethionine. Drug

Fig. 2. Effect of chronic methylcobalamin (10 5 M) exposure on glutamate-induced cytotoxicity. Culture fields were photographed after trypan blue staining followed by formalin fixation. (A) Nontreated cells (control). (B) Cells incubated with glutamate (1 mM) for 10 rain and for a further hour with glutamate-free medium. Cells were maintained in the standard medium. (C) Cells maintained in methylcobalamin-containing medium and treated with glutamate (1 mM). Calibration bar 50 ~zm.

n Viability (%) Control

10 s M

10 7 M

10 6 M

10-5 M

MB12 5 90.0_+0.8 87.7_+1.0 90.0+_0.6 89.0_+0.8 81.7_+2.1 SAM 5 90.7_+0.5 86.8_+0.5 88.0+0.5 87.8_+0.6 88.4+0.7

M i n d u c e d significant c h a n g e s in the viability of the cultures. F i g u r e 2 shows a n example of m e t h y l c o b a l a m i n - i n d u c e d p r o t e c t i o n against g l u t a m a t e cytotoxicity. Most

Protection(%) 0

50 I

I

I

Glutamate ~10~ = 10"8M'TM~ ~

I

!

Protection(%)

100 I

I

[

0

I

~

l

~

Fig. 3. Protective effects of chronic methylcobalaminexposure against glutamate-induced cytotoxicity. Cultures were exposed to glutamate (1 mM) for 10 min, and then incubated in glutamate-free medium for 1 h.

cells under control conditions were not stained by trypan blue (fig. 2A). A 10-min exposure of the cells to glutamate (1 mM), followed by a 1-h incubation, markedly increased the n u m b e r of cells stained by trypan blue (fig. 2B). Chronic administration of methylcobalamin reduced the n u m b e r of cells stained by trypan blue (fig. 2C). Chronic administration of methylcobalamin at 1 0 - 8 - 1 0 -5 M prevented glutamate-induced cytotoxicity in the concentration-dependent manner (fig. 3). Protection by methylcobalamin at 10 -5 M against glutamate cytotoxicity was 64.6 _+ 3.6%. As shown in fig. 4, methylcobalamin (10 5 M) also prevented N M D A - and sodium nitroprusside-induced cytotoxicity. The protection against sodium nitroprussideinduced cytotoxicity was apparently lower than the protection against glutamate- and N M D A - i n d u c e d cytotoxicity. However, there were no statistically significant differences among these values. Figure 5 shows the effects of S-adenosylmethionine on cytotoxicity induced by glutamate and sodium nitroprusside. Chronic administration of 1 0 - s - 1 0 -5 M Sadenosylmethionine prevented glutamate-induced cytotoxicity concentration dependently. The protection by S-adenosylmethionine at 10 -5 M against glutamate cytotoxicity was 61.8_+ 1.2%. S-Adenosylmethionine also prevented the cytotoxicity induced by sodium nitroprusside.

0

I

i

50 I

100

i

'',,

to-SM

~" 10"6 M ~ ~ , ~ , ~

~ 110"5M~

I

=~ 10-8M~ ~ 107M~ 1 0 6 M ~ ~ / ~

Glutamate

:i

I

Protection(%) 50

Sodium

~" to-~Mm ~ tO-~M/

:

nitroprusside

~

1 0 " 6 M ~ "

i

to-~l

I '

Fig. 5. Protective effects of chronic S-adenosylmethionine exposure against cytotoxicity induced by a brief incubation with glutamate (1 mM) or sodium nitroprusside (0.5 mM).

3.3. Effects of acute administration of methylcobalamin and S-adenosylmethionine To examine whether the acute application of methylcobalamin or S-adenosylmethionine with glutamate is effective against glutamate cytotoxicity, the cultures were maintained with the standard medium. Drugs were added to the glutamate-containing medium and to the medium in which the cells were incubated following glutamate exposure. As shown in fig. 6, glutamate cytotoxicity was not prevented by the acute administration of 10 6-10-5 M methylcobalamin. Glutamate cytotoxicity was also not prevented by the acute administration of 10-~'-10 5 M S-adenosylmethionine. In contrast, acute administration of MK-801, a selective N M D A receptor antagonist, potently protected the cells against glutamate cytotoxicity. Protection by the acute administration of 10 5 M MK-801 against glutamate cytotoxicity was 90.3 + 1.2%.

4. Discussion

This study demonstrated that glutamate cytotoxicity was prevented by the chronic administration of methylcobalamin, an active coenzyme of vitamin Bl2 analogs. Glutamate neurotoxicity was also prevented by Sadenosylmethionine, which is formed as an intermedi-

Protection(%) 100

50

o I

I

100 I

I

I

I

eJ 10-6M~}~ I

'utamatel NMoA

I _{ Sodium ~" n troprussde

F

Fig. 4. Protective effects of chronic methylcobalaminexposure against cytotoxicity induced by a brief incubation with glutamate (1 mM), NMDA (1 mM) or sodium nitroprusside (0.5 mM).

Glutamate

Fig. 6. Protective effects of acute methylcobalamin (MB12), Sadenosylmethionine (SAM) and MK-801 exposure against cytotoxicity induced by a brief incubation with glutamate (1 mM).

ate metabolite of methylcobalamin in the metabolic route of vitamin B~z (Hillman, 1990). These results indicate that methylcobalamin possesses neuroprotective effects against glutamate cytotoxicity in cultured rat cortical neurons. Though chronically applied methylcobalamin induced marked protection against glutamate toxicity, acute application of the drug was ineffective. Moreover, methylcobalamin was removed from the glutamate-containing medium between applications in the experiments with chronic drug application. Therefore, it is unlikely that methylcobalamin directly interacts with glutamate receptors. It has been demonstrated that brief glutamate exposure induces delayed death in cultured neurons of the cerebral cortex (Choi et al., 1987). The NMDA subtype of glutamate receptor is considered to be the predominant route of glutamate neurotoxicity, since antagonists of the NMDA receptor prevent neuronal degeneration in cultured cortical neurons. In the present study, NMDA added to the Mg2+-free medium induced cytotoxicity at a magnitude similar to that of glutamate. Moreover, MK-801, a selective blocker of NMDA receptor-gated channels (Young and Fagg, 1990), induces potent protection against glutamate neurotoxicity in cortical cultures maintained in standard medium. These results are consistent with our previous observations (Akaike et al., 1991; Tamura et al., 1992). Therefore, it is conceivable that the cytotoxicity induced by the brief glutamate exposure was mediated by NMDA receptors. Recently, Dawson et al. (1991) have demonstrated, using cortical cultures, that nitric oxide (NO) mediates the NMDA receptor-induced neurotoxicity of glutamate. In our previous study, L-N'-nitro arginine (identical to L-NG-nitro arginine), a NO synthase inhibitor (Moore et al., 1990; Gibson et al., 1990), prevented the neurotoxicity elicited by glutamate or NMDA (Tamura et al., 1992). Moreover, a brief exposure to sodium nitroprusside, which spontaneously releases NO (Katsuki et al., 1977), induced delayed cell death. Cytotoxic effects of nitroprusside were prevented by hemoglobin, which complexes NO. NO, which is apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed in brain tissues. Immunocytochemical studies have demonstrated the presence of NO synthase in select regions of the brain, including the cerebral cortex (Bredt et al., 1990). It has been proposed that NMDA receptor activation causes the formation of NO, which diffuses to adjacent cells, resulting in the appropriate physiological responses a n d / o r glutamate-related cell death. Since cytotoxicity induced by the brief application of sodium nitroprusside is prevented by hemoglobin, it has been accepted that the cytotoxic effect of sodium nitroprusside is mediated by NO (Dawson et al., 1990; Tamura et al., 1992). Therefore, the effects of methyl-

cobalamin on the cytotoxicity induced by sodium nitroprusside were examined to determine whether methylcobalamin protects against the cytotoxicity induced directly by NO. The results of this study showed that chronic administration of methylcobalamin prevented the cytotoxicity induced not only by glutamate but also by sodium nitroprusside. This indicates that the methylcobalamin-treated cultures became tolerant to the cytotoxic effects of NO. Since NO plays a crucial role in NMDA receptor-mediated glutamate toxicity in cortical cultures as mentioned above, it is likely that methylcobalamin-incited prevention of the NO toxicity is the major mechanism of its neuroprotective effects. Involvement of radicals in NO-induced cell injury has been suggested in peripheral tissues (Beckman et al., 1990). Therefore, the NO-related radicals such as hydroxy-free radical (OH') and NO 2 free radical (NO~) may play an important role in the NO-mediated glutamate cytotoxicity observed in the present study, though it is not yet fully elucidated how NO kills neurons in the central nervous system. Thus, we considered that the cultures maintained in the methylcobalamin-containing medium are more tolerant to radical toxicity than those maintained in the standard medium. Chronic treatment of cultures with methylcobalamin may facilitate the elimination of radicals by cellular mechanisms or cause a change in the composition of the cell membrane phospholipids. Methylcobalamin acts as a methyl group donor for the conversion of homocysteine to methionine (Hall, 1990; Hillman 1990). Methionine and its product, S-adenosylmethionine, facilitate intracellular methylation reactions. S-Adenosylmethionine acts as a methyl donor in the reaction forming phosphatidylcholine from phosphatidylethanolamine in the phospholipid layer of the cell membrane. The present study has shown that the chronic administration of S-adenosylmethionine prevented both glutamate- and NO-induced cytotoxicity. Therefore, we concluded that enhanced methylation, such as phosphatidyl choline formation in the membrane phospholipids, is the predominant route of the neuroprotective effect of methylcobalamin. As the neuronal damage cannot be attributed to a single mechanism, there might be other possible influences of methylcobalamin that could be involved in its neuroprotective effects. For example, vitamin B~2 deficiency causes injury to the brain and spinal cord. Hall (1990) postulated that a normally functioning cobalamin-dependent methyl transferase is required for development and function of the brain. Therefore, the methylation metabolism enhanced by the chronic application of methylcobalamin may accelerate neuronal cell growth in the culture. As nerve growth factor reportedly prevents neuronal damage in cortical and hippocampal cell culture (Cheng and Mattson, 1991), methylcobalamin may mimic the effects of nerve growth

factor. The other possible mechanism is reduction of the homocysteine concentration. Homocysteine accumulates in methylcobalamin deficiency (Watkins and Rosenblatt, 1989). Homocysteine is thought to be toxic for the central nervous system (Hall, 1990). Therefore, the mechanism of methylcobalamin-induced neuroprotection may be explained by its effect to decrease the homocysteine level. Although there is no evidence to suggest that chronically applied methylcobalamin reduces receptor expression, we cannot dismiss the effect of methylcobalamin or S-adenosylmethionine on the expression of NMDA receptors in the cultured cortical neurons. However, we estimate that methylcobalaminincited prevention of the NO toxicity is the most important mechanism of its neuroprotective effects since the cytotoxic effects of NMDA and sodium nitroprusside were prevented by methylcobalamin to a similar extent. In conclusion, the present study demonstrate that chronically applied methylcobalamin protects cultured cortical neurons against NMDA receptor-mediated glutamate cytotoxicity. Further studies are necessary to determine the mechanism that is linked to the neuroprotective action of methylcobalamin.

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