Differential effect of β-N-oxalylamino-l-alanine, the Lathyrus sativus neurotoxin, and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate on the excitatory amino acid and taurine levels in the brain of freely moving rats

Abstract

We studied the effect of β-oxalylamino-l-alanine, a glutamate analog present in Lathyrus sativus seeds and implicated in the etiopathogenesis of neurolathyrism, and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate on the extracellular levels of aspartate, glutamate and taurine in the primary motor cortex of freely moving rats. We found that while both neurotoxins increase the level of aspartate and glutamate, only (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate is able to modulate the level of taurine. GYKI-52466, a non-competitive non-NMDA antagonist, inhibited β-oxalylamino-l-alanine-induced increase of aspartate, but not that of glutamate. Conversely, this antagonist proved to be very efficient in blocking the stimulating effect of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate on all three amino acids.

We suggest that β-oxalylamino-l-alanine increases the level of glutamate in vivo by a mechanism not connected to its effect on the non-NMDA receptors, which might involve the inhibition of glutamate transport. This would allow the excitatory neurotransmitter to reach a concentration sufficient to stimulate the non-NMDA receptors, which in their turn mediate the specific release of aspartate. Although the role of aspartate as a neurotransmitter is still under discussion, it might indeed amplify the excitotoxic cascade through its action on NMDA receptors. We speculate that this sequence of events might represent an important step in the molecular cascade leading to the appearance of the selective motoneuron degeneration in neurolathyrism.

Introduction

Neurolathyrism (NL) is a self-limiting neurotoxic disorder, highly prevalent among young males, clinically characterized by irreversible spastic paraparesis (Ludolph et al., 1987, Seyle, 1957, Spencer et al., 1993). Neuropathological studies have demonstrated in NL the presence of degenerative changes in the upper motoneurons (Betz cells) and the corticospinal tract (Streifler et al., 1977).

NL is caused by a continuous dietary consumption (up to 2–4 months) of Lathyrus sativus seeds (chickling pea), a legume resistant to unfavorable environmental conditions (flood or drought), which provides a nourishing and inexpensive component of the daily diet among poor people in certain areas of Asia and Africa (Haimanot et al., 1990, Ludolph et al., 1987). The neurotoxic component of the chickling pea is a glutamate analog named β-N-oxalylamino-l-alanine (BOAA). BOAA shows some structural similarity to another non-protein amino acid, known as β-N-methylamino-l-alanine (BMAA), which has been found in the seeds of Cycas circinalis (Spencer, 1990). BMAA was initially suggested as being the responsible agent for the Western Pacific amyotrophic lateral sclerosis/parkinson-dementia complex found in the Chamorros of the Mariana Islands (Kurland, 1988, Spencer et al., 1987). However, while a cause-to-effect relationship between BMAA exposure and the Chamorros amyotrophic lateral sclerosis/parkinsonism-dementia complex was not proved (Duncan et al., 1990), it is now clearly established that BOAA exposure leads to NL (Ludolph and Spencer, 1996). Interestingly, it has been reported that some of the patients with a long-lasting NL developed a clinical course resembling that of amyotrophic lateral sclerosis (ALS), with progressive worsening of the symptomatology and involvement of lower (spinal) motoneurons (Cohn and Streifler, 1981, Ludolph et al., 1987). This evidence suggests that yet-to-be discovered environmental factors might be related, directly or indirectly, to the pathogenesis of ALS.

The mechanism of BOAA-induced neurotoxicity is still not completely understood. Previous in vitro studies have shown that BOAA binds to α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (Ross et al., 1989, Sawutz et al., 1995), and inhibits glutamate uptake in a synaptosomal preparation (Ross et al., 1985). When administered to rats at non-convulsant doses, BOAA increases cerebellar cyclic GMP, an effect blocked by the non-selective glutamate antagonist kynurenate, and induces a down-regulation of glutamate receptors in the primary motor cortex (La Bella et al., 1993a, La Bella et al., 1993b). Taken together, these experiments suggest that BOAA might induce neuronal degeneration through an excessive stimulation of glutamate receptors. There are, however, remarkable species differences in susceptibility to the neurotoxin: whereas chicks are readily susceptible to BOAA, rats or mice do not develop neurodegenerative changes (Rao, 1978, Spencer et al., 1991), though behavioral changes reminiscent of an excitable status can be reproducibly detected (La Bella et al., 1997).

Recently, several alternative mechanisms to explain BOAA toxicity have been suggested. There are evidences that the chickling pea neurotoxin can modulate the activity of some enzymes, such as the astrocyte glutamine synthetase (induction) (Miller et al., 1993) and the tyrosine aminotransferase (inhibition) (Vardhan et al., 1997). More controversial is its effect on NADH-dehydrogenase: an initial study reported an inhibitory effect of very low doses of BOAA on this enzyme (Pai and Ravindranath, 1993), but these results were not confirmed in another study (Sabri et al., 1995). Another interesting putative mechanism of BOAA toxicity, which would indirectly reconcile to the excitotoxic theory, is given by its effect on the Xc cystine/glutamate antiporter. BOAA, in fact, induces cell toxicity through a block of the Xc antiporter (Murphy et al., 1990), which ultimately leads to apoptotic cell death (La Bella et al., 1996). Taken together, these studies indicate that the mechanism of BOAA-mediated neuronal degeneration cannot be explained solely on the basis of a non-NMDA (AMPA) receptor-mediated toxicity. This hypothesis is supported by a study in which BOAA-induced, but not AMPA-induced, neurotoxicity following focal hippocampal injection was prevented by free radical scavengers (Willis et al., 1994).

Using a microdialysis approach in the present study we have investigated the effect of the local infusion of the chickling pea neurotoxin and AMPA on the extracellular levels of some amino acid neurotransmitters (aspartate, glutamate, and taurine) in the primary motor cortex and dorso-lateral striatum of freely-moving rats. We show here that both neurotoxins induce a transient increase of aspartate and glutamate, whereas only AMPA is able to modulate the level of taurine. Moreover, non-NMDA antagonists blocked AMPA-mediated, but not BOAA-mediated, glutamate increase. These results strongly suggest the possible existence of different pathways for the neurotoxic effects of BOAA and AMPA in vivo.