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The cycad neurotoxic amino acid, ß-N -methylamino-L-alanine(BMAA), elevates intracellular calcium levels in dissociated rat brain

cells

Delia M. Brownsona,d, Tom J. Mabry a,b,d,*, Steven W. Leslie c,d

a Department of Botany, School of Biological Sciences, The Uni versity of Texas at Austin, Austin, TX 78712, USAb Molecular Cell and Developmental Biology, The Uni versity of Texas at Austin, Austin, TX 78712, USA

c Di vision of Pharmacology and Toxicology, College of Pharmacy, The Uni versity of Texas at Austin, Austin, TX 78712, USAd Institute for Neuroscience, The Uni versity of Texas at Austin, Austin, TX 78712, USA

Received 1 November 2001; received in revised form 1 April 2002; accepted 20 June 2002

Abstract

Seeds of the Guam cycad Cycas micronesica K.D. Hill (Cycadaceae), which contain ß-methylamino-L-alanine (BMAA), have

been implicated in the etiology of the devastating neurodisease ALS Á /PDC that is found among the native Chamorros on Guam.

The disease also occurs in the native populations on Irian Jaya and the Kii Peninsula of Japan, and in all three areas the cycad seeds

are used either dietarily or medically. ALS Á /PDC is a complex of amyotrophic lateral sclerosis and parkinsonism dementia complex

with additional symptoms of Alzheimer’s. It is well known that Ca2' elevations in brain cells can lead to cell death and

neurodiseases. Therefore, we evaluated the ability of the cycad toxin BMAA to elevate the intracellular calcium concentration

([Ca2']i ) in dissociated newborn rat brain cells loaded with fura-2 dye. BMAA produced an increase in intracellular calcium levels

in a concentration-dependent manner. The increases were dependent not only on extracellular calcium concentrations, but alsosignificantly on the presence of bicarbonate ion. Increasing concentrations of sodium bicarbonate resulted in a potentiation of the

BMAA-induced [Ca2']i elevation. The bicarbonate dependence did not result from the increased sodium concentration or

alkalinization of the buffer. Our results support the hypothesis that the neurotoxicity of BMAA is due to an excitotoxic mechanism,

involving elevated intracellular calcium levels and bicarbonate. Furthermore, since BMAA alone produced no increase in Ca2'

levels, these results suggest the involvement of a product of BMAA and CO2, namely a b-carbamate, which has a structure similar to

other excitatory amino acids (EAA) such as glutamate; thus, the causative agent for ALS Á /PDC on Guam and elsewhere may be the

b-carbamate of BMAA. These findings support the theory that some forms of other neurodiseases may also in volve environmental

toxins. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: ß-N -Methylamino-L-alanine; Excitatory amino acids; Neurotoxicity; Fura-2 dye; Calcium; Amyotrophic lateral sclerosis Á /parkinsonism

dementia complex, BMAA-carbamate

1. Introduction

The high incidence of amyotrophic lateral sclerosis Á /

parkinsonism dementia complex (ALS Á /PDC) among

the Chamorro population on Guam following World

War II has been linked by several investigators to the

dietary and medicinal use of seeds of the Guam cycad

(Whiting, 1963; Kurland, 1972; Spencer et al., 1987a);

the Guam cycad is now referred to as Cycas micronesica

K.D. Hill (Cycadaceae) (Hill, 1994, 1995). Although

ALS Á /PDC occurs in several genetically and geographi-

cally distinct ethnic groups in the western Pacific,

namely the indigenous populations of the Mariana

Islands, (Guam, Rota), Irian Jaya and the Kii Peninsula

of Japan, it has been most studied among the Chamor-

ros on Guam where the incidence of the disease is 100

times higher than in the mainland US (Kurland, 1972;

Spencer et al., 1987a,b,c; McGeer et al., 1997). Some

Chamorros patients manifest signs of parkinsonism

characterized by slowed movements, tremor, and rigid-

* Corresponding author. Tel.: '/1-512-417-1900; fax: '/1-512-232-

3402

E-mail address: [email protected] (T.J. Mabry).

Journal of Ethnopharmacology 82 (2002) 159 Á /167

www.elsevier.com/locate/jethpharm

0378-8741/02/$ - see front matter# 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 1 7 0 - 8

mailto:[email protected]

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ity, while others exhibit the progressive limb weakness

common with amyotrophic lateral sclerosis (ALS). Still

other patients develop cognitive dysfunction typical of

dementia found in patients with Alzheimer’s. Epidemio-

logical studies strongly suggest that the Guam disease

represents neurological damage caused by some envir-

onmental factor such as the toxin in cycad seeds(Kurland, 1972).

The Chamorros were aware that the cycad seeds were

toxic and generally washed the dried seed ‘chips’ used to

prepare a flour with unheated water multiple times over

several days, a procedure that should remove most of

both the toxic cycasin (methylazoxymethanol b-D-gluco-

side) as well as the ß-methylaminoalanine (BMAA), the

compound investigated here (Kurland, 1972; Duncan et

al., 1990). Some researchers have argued that there is

just too little BMAA remaining in the washed cycad

flour to account for the ALS Á /PDC on Guam (Duncan

et al., 1990). Therefore, a recent paper by Cox and Sacks(2002) is of considerable interest; they suggest that flying

foxes (bats) may have been an important source of the

cycad toxin for the Chamorros. Bats can triple their

weight in just one evening of feeding on cycad seeds, and

the natives would regularly feast on these animals when

cooked in coconut cream, fur and all. Significantly,

flying fox consumption was confined to the Chamorros,

the very group that experienced the highest known

incidence of ALS Á /PDC (Cox and Sacks, 2002). More-

over, over the last 40 years, the occurrence of ALS Á /

PDC among the Chamorros has not only declined in a

manner consistent with the decreased traditional dietary

use of the cycad seeds as a source of starch (Kurland,1972; Stone, 1993) but also with the near disappearance

by the 1970’s of the Guam flying fox population due to

over-hunting (Cox and Sacks, 2002). In two other

locations where ALS Á /PDC occurs, Irian Jaya and the

Kii Peninsula of Japan, raw seeds of the Cycas revoluta

Thunb. are used by natives as topical medicines for cuts,

hemorrhoids, and open sores (Spencer et al., 1987a,b,c),

and we recently found that C. revoluta seeds also

contain the Guam cycad toxin BMAA (Pan et al.,

1997a,b). Thus, ALS Á /PDC may be an example of a

chronic neurodegenerative disease induced by exposure

to excitotoxins of exogenous origin, in this case possiblythe non-protein amino acid ß-N -methylamino-L-alanine

(BMAA) present in seeds of C. micronesica (Vega and

Bell, 1967). Therefore, since elevations in Ca2' levels in

brains can lead to cell death and neurodiseases, we

investigated under what conditions BMAA could raise

Ca2' levels in brain cells of newborn rats.

Results from several studies indicate that BMAA may

mediate neurodegeneration via an excitotoxic mechan-

ism induced by the activation of excitatory amino acid

(EAA) receptors. Repeated oral administration of sub-

convulsant doses of BMAA to macaques produced a

neurological syndrome with features of ALS Á /PDC

(Spencer et al., 1987a). In young rats, the intraperitoneal

administration of high doses of BMAA produced

abnormal movements and selective neuronal cell death

in the cerebellum (Seawright et al., 1990). Acute con-

vulsant effects of BMAA have been reported to be

attenuated by antagonists of either NMDA (N -methyl-

D-aspartate) or non-NMDA receptors (Ross and Spen-cer, 1987; Smith and Meldrum, 1990). In spinal cord

cultures, BMAA produced neuronal vacuolation and

death (Nunn et al., 1987). At high concentrations,

BMAA-induced neuronal degeneration was attenuated

by NMDA receptor antagonists and open-channel

blockers (Ross and Spencer, 1987; Ross et al., 1987;

Spencer et al., 1987a; Weiss and Choi, 1988; Weiss et al.,

1989; Zeevalk and Nicklas, 1989). BMAA administered

neonatally to rats, produced permanent developmental

damage to motor function and spinal cord neurochem-

istry (Dawson et al., 1998).

A requirement of extracellular bicarbonate has beendocumented for the neurotoxic effects of BMAA in

cultured cortical neurons (Weiss and Choi, 1988; Weiss

et al., 1989), isolated chick retina (Zeevalk and Nicklas,

1989) and spinal cords (Stewart et al., 1991). The

bicarbonate dependency may relate to the formation

of a BMAA-carbamate that has a structure similar to

other EAAs (Nunn and O’Brien, 1989; Myers and

Nelson, 1990). Electrophysiological studies have demon-

strated that the presence of NaHCO3 greatly potentiates

BMAA-mediated conductance. BMAA-mediated cur-

rents are blocked by non-NMDA receptor antagonists

(Allen et al., 1993, 1995).

Neurochemical and binding studies indicate thatBMAA is a mixed EAA receptor agonist. BMAA

inhibited [3H]-glutamate binding to the metabotropic

site (Cha et al., 1990) and the ability to displace

specifically bound [3H]-glutamate was greater in the

presence of 25 mM bicarbonate (Richter and Mena,

1989; Copani et al., 1991). Strychnine-insensitive [3H]-

glycine binding was inhibited by BMAA; however,

BMAA had very low affinity for NMDA receptors

that was displayed only in the presence of bicarbonate

(Copani et al., 1991). BMAA strongly stimulated

polyphosphoinositol hydrolysis that was blocked by

selective antagonism of metabotropic receptors (Copaniet al., 1990, 1991). BMAA induced necrosis and

apoptosis of rat cerebellar granule cells via group III

metabotropic receptors (Staton and Bristow, 1997,

1998). Thus, BMAA may most potently activate the

metabotropic receptor subtype.

A central tenet of the excitotoxicity hypothesis

suggests that exposure to EAAs causes excessive in-

tracellular calcium ion accumulation, which may act as a

trigger for the neurodegenerative process (Rothman and

Olney, 1987; Choi, 1988; Frandsen and Schousboe,

1993). Considerable experimental evidence indicates

that Ca2' influx is essential to EAA-induced neuro-

D.M. Brownson et al. / Journal of Ethnopharmacology 82 (2002) 159 Á / 167 160

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toxicity (Choi, 1987; Randall and Thayer, 1992; Ty-

mianski et al., 1993). Sustained impairment of intracel-

lular calcium homeostasis is thought to cause disruption

of the Ca2'-dependent cascades responsible for neuro-

nal death (Choi, 1988; Meldrum and Garthwaite, 1990).

Recently, the mechanism of Ca2' entry was demon-

strated to be a more important determinant of EAA-induced neurotoxicity than peak or average intracellular

Ca2' elevations (Tymianski et al., 1993).

The ability of BMAA to increase intracellular calcium

levels has not been previously demonstrated. To deter-

mine whether intracellular calcium changes in response

to BMAA could be monitored, dissociated rat brain

cells were loaded with the fluorescent dye fura-2 (Fig. 1);

the dye forms a spectrally measurable Ca2'-complex

(Fig. 2) allowing determination of the Ca2' levels in the

rat brain cells. BMAA-mediated responses in relation to

the extracellular calcium concentration and the presence

of bicarbonate were also determined; other factors of excitotoxicity have been presented elsewhere (Brownson

et al., 1993; Brownson, 1996).

2. Materials and methods

2.1. Chemicals

Fura-2 dye (fura-2 acetoxymethyl ester) was pur-

chased from Molecular Probes (Eugene, OR); BMAAfrom Research Biochemicals Inc. (Natick, MA); plasma-

derived horse serum and trypsin from Gibco (Grand

Island, NY); deoxyribonuclease I from Boehringer

Mannheim (Indianapolis, IN); Dulbecco’s modified

Eagle’s Medium, NMDA and other chemicals from

Sigma Chemical Co. (St. Louis, MO).

2.2. Isolation of dissociated rat brain cells and fura-2

loading into cells

The Animal Resources Center at the University of

Texas at Austin maintains a breeding colony of

Sprague Á /Dawley rats. Whole brain cells from neonatal

(B/24 h) rats from the Animal Center were prepared

according to the method of Dildy-Mayfield and Leslie

(1991). Five animals per experiment were decapitated,

brain tissue was removed and kept in ice-cold balanced

salt solution (BSS; containing in mM: NaCl, 137; KCl,

5.4; Na2HPO4, 0.17; KH2PO4, 0.22; glucose, 5.5,

sucrose, 59; phenol red, 0.03; pH held at 7.4 with

NaOH) until dissections were complete. Tissue was

transferred to a small glass Petri dish on ice where the

meninges were carefully removed, and the tissue was

finely minced. The minced tissue was rinsed in BSS,

placed in a trypsinization flask and dissociated with 8 mlof 0.05% trypsin and 0.5 mM EDTA in BSS, while

gently stirring (250 rpm) for 10 min and carefully

monitoring the temperature at 37 8C and pH at 7.4.

Next, the tissue was allowed to settle, and the disso-

ciated cell suspension was transferred to a second

trypsinization flask containing 9 ml of Dulbecco’s

modified Eagle’s medium (high glucose, 4.5 g/l) with

10% (v/v) plasma-derived horse serum and 3.3 mg/ml

DNase, warmed to 37 8C. The tissue was stirred gently

(175 rpm) in a 37 8C water bath for 5 min. The

dissociated cell suspension was collected to exclude

small tissue fragments, divided equally into two centri-fuge tubes, and centrifuged at 300)/ g for 5 min. The

supernatants were decanted and the pellets were resus-

pended in 4 ml of HEPES-buffered Hanks’ solution

(HBH) (containing in mM: HEPES, 20; NaCl, 137;

CaCl2, 1.3; KCl, 5.0; Na2HPO4, 0.6; KH2PO4, 0.4;

NaHCO3, 3.0; MgSO4, 0.4; MgCl2, 0.5; glucose, 5.6; pH

to 7.4 with NaOH). The cell density (via hemacytometer

count) and viability (via exclusion of trypan blue) of the

cell suspension were then determined. An additional 4

ml of HBH were added, and the cell suspension was

divided equally into two aliquots. To one aliquot, 20 ml

of 5 mM fura-2-acetoxymethyl ester (fura-2 dye) dis-

Fig. 1. The experimental procedures employed for determining if

BMAA can elevate Ca2' levels in rat brain cells were as follows: the

cells were first soaked in a solution containing fura-2 dye; next, the

dye-loaded cells were placed in either a Ca2'-BMAA solution or one

containing Ca2', BMAA, and bicarbonate. Finally, in both experi-

ments the amount of fura-2-Ca2' complex formed inside the cells was

spectrally measured.

Fig. 2. Fura-2 dye complexed with Ca2'; the complex gives a readily

measured fluorescent spectrum.


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solved in DMSO were added. To the other aliquot, 20 ml

of DMSO were added and treated in parallel to serve as

a control for autofluorescence (non-loaded cells). The

cell suspensions were incubated at 37 8C in a shaking

water bath. After 1 h, both fura-2-loaded and non-

loaded cells were separately collected and centrifuged at

300)/ g for 5 min, and resuspended in an appropriatevolume of Mg2'-free medium to give a suspension of

3.5)/106 cells per 2 ml aliquot. For particular experi-

ments, the resuspension media were modified as detailed

below. These cell suspensions were presumed to be

viable during the next hour when the experiments were

conducted.

2.3. Fluorescence measurements

Both fura-2-loaded and non-loaded cell suspensions

were kept at room temperature until fluorescence

measurements were completed. Fluorescence measure-ments and calculations of calcium levels were

determined using a SPEX Fluorolog spectrophoto-

fluorometer (Model CM2 Cation Measurement

System). Aliquots (2 ml) of the fura-2-loaded cell

suspension were incubated at 37 8C for 2 min, then

placed in the cuvette compartment of the SPEX instru-

ment and continuously mixed with a magnetic stirrer.

Fluorescence scans were acquired using dual monochro-

maters with 340 and 380 nm excitation and monitoring,

respectively, and 505 nm emission. Fluorescence inten-

sity was measured every second, and averages were

recorded every 3 s for 180 s. Basal fluorescence intensity

was measured during the first 51 s of each time scan toestablish resting intracellular calcium levels. Unless

otherwise stated, BMAA was dissolved in 1 M

NaHCO3. After 51 s of the initial fluorescence measure-

ment, BMAA (20 ml) was added. During the last 15 s of

each fluorescence scan, 40 mM MnCl2 (50 ml of 1.64 mM

MnCl2) were added to the sample. Since Mn2'

quenches extracellular fura-2 fluorescence, the net

decrease in fluorescence intensity at 340 nm (i.e. the

Mn2' correction) was subtracted from the entire 340

nm time scan to account for dye leakage of individual

samples.

2.4. Fluorescence parameters and experimental

calculations

The intracellular free calcium concentration ([Ca2']i )

was determined according to the method of Grynkiewicz

et al. (1985) using a Kd of 224 nM; [Ca2']i 0/(Kd)Sf2/

Sb2 (R(/Rmin/Rmax)/R ). For each preparation, the

fluorescence parameters Sf2/Sb2, Rmax and Rmin were

determined from excitation scans (330 Á /390 nm) of two

samples of fura-2-loaded cells, lysed with 0.1% sodium

dodecyl sulfate and containing either 4 mM CaCl2 (max)

or 5 mM EGTA in 3 M Tris base (min). Background

fluorescence at 340 and 380 nm of two non-loaded

samples were treated in a similar manner and the

findings were subtracted from the excitation scans of

the fura-2-loaded cells prior to ratio calculations. An

additional excitation scan of untreated non-loaded cells

provided an estimate of autofluorescence at 340 and 380

nm; these results were then subtracted from each sampleprior to further analysis. The R value was equal to the

ratio of 340/380 nm fluorescence intensity at each time

point of the individual fluorescence scans.

2.5. Cell suspension media

The fura-2-loaded and non-loaded cells were resus-

pended in media selected according to the specific

conditions and aims of the particular experiments. For

initial experiments designed to obtain a concentration Á /

response curve for BMAA, cells were resuspended in

Mg2'

-free HBH. Another concentration Á /responsecurve of BMAA was determined at both a constant

pH and constant bicarbonate ion concentration through

the use of a bicarbonate-based buffer (BicBH). For the

BicBH, 24 mM sodium bicarbonate were substituted for

the 20 mM HEPES, with other ingredients of the

buffered Hank’s medium unaltered. The BicBH was

then equilibrated with a gas mixture of 5% CO2/95% O2

to pH 7.4 at 37 8C. The cell pellet was resuspended in

BicBH and kept under 5% CO2/95% O2 throughout the

experiment, including the period of fluorescence mea-

surements. To determine the involvement of extracellu-

lar calcium, cells were resuspended in HBH that

contained no added CaCl2 (nominally calcium-free).Various calcium concentrations (0, 0.08, 0.16, 0.32, 0.65,

1.3, and 2.6 mM) were then added (20 ml stock into 2 ml

aliquots) during the incubation period just prior to

fluorescence measurements.

Two series of experiments were conducted to confirm

that the ability of BMAA to increase Ca2' levels was

dependent on the presence of bicarbonate. In these

experiments, 5 mM BMAA was dissolved in HBH

medium without MgCl2 or with NaHCO3. Cells were

initially resuspended in Mg2'-free HBH to one-fifth

volume calculated to give a suspension of 3.5)/106 cells

per 2 ml aliquot. The cells were then diluted theremaining four-fifths volume with various desired

media. In the first set of experiments, cell suspensions

were exposed to a range of NaHCO3 concentrations (3,

5.4, 10.2, 19.8 and 39 mM). Since pH and sodium

concentration varied with addition of NaHCO3, a

second set of experiments was performed as a control

to demonstrate the effect of the bicarbonate. Cell

suspensions containing 3 or 19.8 mM NaHCO3 were

prepared as above. Two additional samples were

brought up to the final volume with Mg2'-free HBH

media that was either alkalinized or contained added

NaCl to match that of the 19.8 mM NaHCO3 samples


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above. The calibration of fluorescent signals was not

noticeably affected by the various resuspension media.

For most experiments, statistical analyses were per-

formed using analysis of variance, and BMAA con-

centration Á /response curves were predicted using a four-

parameter (a0/maximum, b0/slope, c0/EC50 50 and

d0/minimum) logistic equation using ALLFIT (DeLean et al., 1978; Brownson, 1996).

3. Results

The findings described here suggest a mechanism by

which BMAA, the cycad toxin, might be a factor in

ALS Á /PDC among the Chamorros on Guam. In dis-

sociated rat brain cell preparations, BMAA produced

nanomolar increases of intracellular calcium concentra-

tions ([Ca2']i ) in the presence of bicarbonate. Fig. 3

(top trace) shows the results of a typical experiment inwhich the change of [Ca2']i was monitored over time

with the addition of 5 mM BMAA at approximately 51

s. Addition of BMAA dissolved in NaHCO3 caused an

increase of [Ca2']i that reached peak levels within 20 s,

but partially declined in a manner similar to that

reported with 100 mM quisqualate (Dildy-Mayfield et

al., 1991). The magnitude of these [Ca2']i increases

were similar to those previously observed in dissociated

neurons with the addition of 100 mM quisqualate or 100

mM kainate but appeared to be less than that of 25 mM

NMDA (Dildy-Mayfield and Leslie, 1991; Dildy-May-

field et al., 1991). Addition of BMAA dissolv

ed in watershowed little ability to increase [Ca2']i (Fig. 3, middle

trace) and 10 mM NaHCO3 alone caused only a slight

decrease in [Ca2']i (Fig. 3, bottom trace).

The resting [Ca2']i (averaged from 0 to 51 s) were

typically within a 150 Á /180 nM range for cells resus-

pended in HBH buffer containing various concentra-

tions of NaHCO3 as well as in the BicBH buffer. These

resting [Ca2']i were similar to those reported by others

using dissociated brain cell preparations (Dildy-May-

field and Leslie, 1991; Dildy-Mayfield et al., 1991).

Thus, resting [Ca2']i were not affected by the various

resuspension media; however, the initial resting [Ca2']i

did depend on the extracellular CaCl2 concentration.

Reported D [Ca2']i values were calculated as the

difference between the [Ca2']i (averaged over 51 s after

addition of BMAA or other materials) and initial resting

[Ca2']i (averaged from 0 to 51 s).

The effect of increasing concentrations of BMAA was

determined both in HBH buffer, typically used in the

dissociated brain cell preparations (Dildy-Mayfield and

Leslie, 1991), and in BicBH, where bicarbonate-CO2

replaced the buffering capacity of HEPES. The compu-

ter program ALLFIT (De Lean et al., 1978) was used to

predict and statistically test sigmoidal concentration Á /response curves for BMAA. These statistical analyses

indicated that the BMAA concentration Á /response

curves were parallel but had different EC50 values,

with those in BicBH medium shifted to the left relative

to those obtained in a solution buffered with HEPES.

The extracellular calcium dependency of BMAA-

mediated responses was examined. Initially, cells were

resuspended in HBH medium without added MgCl2 or

CaCl2. Next, various concentrations of CaCl2 (0 Á /2600

mM) were added to samples during the 2 min incubation

period prior to fluorescence measurements. Subse-

quently, resting [Ca2']i of samples were stable. To

give a consistent and substantial response, the concen-tration of 5 mM BMAA (dissolved in 10 mM NaHCO3)

was selected. In suspensions nominally calcium-free (i.e.

no added CaCl2), the resting [Ca2']i was 51.79/5.6 nM

and BMAA caused an apparent decrease in [Ca2']i of

16.4 nM. As greater amounts of CaCl2 (0.08, 0.16, 0.32,

0.65, 1.3, and 2.6 mM) were added, the magnitude of

initial resting [Ca2']i also increased (values of 56.19/

1.5; 65.29/2.1; 79.49/3.6; 96.79/6.3; 128.39/6.8; 179.39/

12.4 nM, respectively). BMAA raised [Ca2']i above

resting levels at higher extracellular calcium concentra-

tions. Thus, the magnitude of BMAA-mediated [Ca2']i

increases were significantly dependent on extracellularcalcium levels.

Two additional sets of experiments were performed to

further characterize the dependence of the BMAA

response on the presence of the bicarbonate ion. In the

first set of experiments, both fura-2-loaded and non-

fura-2-loaded cells were resuspended in Mg2'-free HBH

buffer containing varying concentrations of NaHCO3.

With increasing NaHCO3 concentrations, the pH of the

medium became more alkaline; however, the pH was not

adjusted. Fluorescence parameters were determined

separately for each suspension treatment. It was clearly

evident that the BMAA-mediated [Ca2']i increases in

Fig. 3. Brownson showed that BMAA increased Ca2' levels in the rat

brain cells but only in the presence of bicarbonate implicating the

carbamate of BMAA, a derivative that is structurally similar to

glutamic acid.


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the rat brain cells were highly dependent on the presence

of bicarbonate (3 Á /40 mM) (Brownson, 1996).

Additional control experiments were performed to

demonstrate that the ability of BMAA to elevate

[Ca2']i was dependent on the presence of bicarbonate

ion and not on the alkalinization of the medium or the

increases in the sodium concentration. Both fura-2-loaded and non-fura-2-loaded cells were resuspended in

HBH buffer containing either additional NaHCO3 (19.2

mM), NaCl (19.2 mM), or NaOH (pH was adjusted to

match the alkalinization of the 19.2 mM NaHCO3

sample). Again, fluorescence parameters were deter-

mined separately for each suspension treatment.

BMAA did not significantly increase [Ca2']i of dis-

sociated cells in either alkaline or NaCl-containing

media relative to those resuspended in HBH (control).

4. Discussion

EAAs have been implicated in both acute and chronic

neurological disorders (Rothman and Olney, 1987;

Choi, 1988; Meldrum and Garthwaite, 1990; Meldrum,

1993). Two known excitotoxins are of dietary origin: (a)

ß-N -oxalylamino-L-alanine (BOAA), which has been

established as the cause of neurolathyrism associated

with excess consumption of chickling pea (Lathyrus

sati vus L., Leguminosae) (Spencer et al., 1986; Bridges

et al., 1989) and (b) domoate from diatoms, which

causes toxic encephalopathy due to ingestion of con-

tamined mussels (Perl et al., 1990; Teitelbaum et al.,

1990). The Guam ALS Á /PDC may be an example of achronic neurodegenerative disease induced by exogen-

ous EAA-exposure (Meldrum, 1993), possibly due to the

medicinal and dietary use of cycad seeds (Whiting, 1963;

Kurland, 1972; Spencer et al., 1987a,b,c; Smith and

Meldrum, 1990). The non-protein amino acid BMAA is

present in cycad seeds (Vega and Bell, 1967) and has

been implicated as an etiological factor by animal

models (Spencer et al., 1987a). BMAA can induce

neurotoxicity, presumably by an excitotoxic mechanism

since neuronal cell death can be prevented by both

NMDA and non-NMDA receptor antagonists (Weiss

and Choi, 1988; Weiss et al., 1989). Based on neuro-chemical and binding studies, Copani et al. (1991)

suggested that BMAA most likely activates non-

NMDA receptors, especially at lower concentrations.

The objective of this study was to determine whether

dissociated newborn rat brain cells loaded with the

fluorescent fura-2 dye (Figs. 1 and 2) could be utilized to

monitor the effect of BMAA on [Ca2']i . Calcium influx

is thought to be intimately involved in EAA-induced

neurotoxicity. A sustained impairment of intracellular

calcium homeostasis may lead to deleterious activation

of several Ca2'-dependent enzymes implicated in the

pathophysiology of neuronal damage (Choi, 1988;

Meldrum and Garthwaite, 1990). Copani et al. (1991)

reported that BMAA in the presence of 1 mM NaHCO3

did not increase 45Ca2' uptake but reversed kynurenic

acid-inhibition of glutamate-stimulated 45Ca2' influx.

The work presented here demonstrates that BMAA in

the presence of bicarbonate raises [Ca2']i in a concen-

tration-dependent manner (Fig. 3) and supports thehypothesis that the action of BMAA is similar to other

excitotoxins and involves the activation of EAA recep-

tors and sustained [Ca2']i accumulation.

BMAA is reported to be a potent activator of the

metabotropic receptor coupled to polyphosphoinositide

hydrolysis (Copani et al., 1991), which can subsequently

release Ca2' from intracellular sources (Berridge, 1993;

Frandsen and Schousboe, 1993). Since the BMAA-

mediated [Ca2']i increases reported here might be due

to intracellular release of Ca2', the effects of varying

extracellular calcium concentrations on BMAA-induced

responses were examined. With cells resuspended inHBH that contained less than 162.5 mM CaCl2, BMAA

produced a small decrease in [Ca2']i compared with

initial resting levels. This is similar to the effect observed

with reduced glutathione in the dissociated cell prepara-

tions (Leslie et al., 1992). The significance of this latter

finding is unclear, but most likely relates to effects of

omission of CaCl2 from the medium and low resting

[Ca2']i . As greater amounts of CaCl2 were added,

resting [Ca2']i increased and the magnitude of

[Ca2']i elevation mediated by BMAA increased. These

results suggest that increases of [Ca2']i by BMAA were

due to extracellular Ca2'

entry. Our study, however,cannot preclude the possibility that BMAA may also

liberate Ca2' from intracellular sources, although we

have established that such a release would have to be

triggered by Ca2' influx (Berridge, 1993; Frandsen and

Schousboe, 1993).

Significantly, the ability of BMAA to elevate [Ca2']i

was critically dependent on the presence of the bicarbo-

nate ion. Previously, Weiss and Choi (1988) noted

differences in the ability of BMAA to cause neurode-

generation depending on the culture medium used.

These researchers documented a unique requirement of

physiological extracellular bicarbonate for the expres-sion of BMAA-induced neurotoxicity. Similarly, Copani

et al. (1991) reported a bicarbonate dependency for

BMAA in established neurochemical correlates of EAA

receptor activation such as in polyphosphoinositide

hydrolysis and in EAA receptor binding studies.

BMAA does not have all the structural characteristics

of EAA agonists; however, in the presence of bicarbo-

nate a BMAA Á /carbamate adduct is formed (Nunn and

O’Brien, 1989; Myers and Nelson, 1990). Thus, interac-

tion between BMAA and bicarbonate ions provides a

molecular configuration that may activate EAA recep-

tors, and therefore, be responsible for the ability of


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BMAA in buffers containing bicarbonate to elevate

Ca2' levels in rat brain cells.

Our results are consistent with the hypothesis that an

interaction between BMAA and the bicarbonate ion

produces a new agonist species, namely, the ß-carba-

mate of BMAA (Fig. 4), which is almost isosteric with

glutamate (Myers and Nelson, 1990) and is most likelyresponsible for the EAA-like activity of BMAA in the

presence of bicarbonate. The importance of such a

mechanism is suggested since no elevations of [Ca2']i

were observed in the absence of bicarbonate. Also, the

magnitude of BMAA-mediated increases are directly

dependent on the concentration of bicarbonate ions but

are not due to increases in pH or sodium ions.

The potential implications of our results regarding the

role of the carbamate of BMAA in the etiology and

pathology of ALS Á /PDC are difficult to assess. Some

researchers suggest that involvement of BMAA in this

chronic neurodegenerative disease seems improbable, inpart, because BMAA generally lacks the potency of

other acute excitotoxins, and because large quantities of

BMAA were required to induce a neurological syn-

drome in macaques that resembles the Guam ALS Á /

PDC (Spencer et al., 1987a). In addition, the small

amounts of BMAA present in seeds of C. micronesica

may be removed when the seeds are thoroughly washed

with water prior to preparation of the flour (Duncan et

al., 1990; Kisby et al., 1992). However, it has now been

suggested that flying foxes (bats) on Guam may have

been a major source of BMAA for the Chamorros who

regularly feasted on bats that fed on cycad seeds (Cox

and Sacks, 2002). Moreover, the decline by the 1970’s of

both ALS Á /PDC and the bat population on Guam is

closely correlated (Cox and Sacks, 2002). The amount of

BMAA Á /b-carbamate in a bicarbonate solution of

BMAA under the conditions in our experiments (and

under physiological conditions) is about 10% in an

equilibrium reaction as estimated by NMR studies

(Myers and Nelson, 1990). The detailed ionic and

possibly cyclic structure (involving the a-amine) of the

BMAA Á /b-carbamate and its interaction with neuror-

eceptors will be further investigated. Other compounds

such as cysteine, which by themselves are not structu-

rally recognizable as EAA agonists, but may also form

carbamates and induce neuronal injury by an excitotoxic

mechanism (Olney et al., 1990). The fact that BMAA

requires physiological concentrations of bicarbonate toinduce elevations of [Ca2']i in rat brain cells may have

several important implications for chronic excitotoxicity

and pathogenesis of ALS Á /PDC. Thus, the findings

described here for BMAA and its b-carbamate further

implicate the dietary (perhaps via flying foxes, fur and

all, cooked in coconut sauce) of BMAA-containing

cycad seeds on the Island of Guam, and medicinal use

elsewhere, as a causative factor in ALS Á /PDC. Our

findings for the carbamate of BMAA and ALS Á /PDC

among the Chamorros on Guam support the theory that

environmental toxins cause some forms of Parkinson’s,

Alzheimer’s, and ALS as well as the Gulf War Syn-drome.

Acknowledgements

The authors are grateful for support by National

Institute on Aging (NIH: Grant R01 AG 10637), NATO

(Grant CRG 910429), NIAAA (Grant R37 AA05809),

and The Robert A. Welch Foundation (Grant F-130).

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