In Sec. III we reviewed the evidence showing that glutamate toxicity is mediated by NMDA glutamate receptors which, when activated, lead to Ca2+ entry which, in turn, activates the production of NO by NOS. Thus, it is the increase in NO concentration in the cell that would be responsible for many of the toxic effects of glutamate (6). On the other hand, recent observations also show that NO donors and peroxynitrite (ONOO~) stimulate a sustained increase of [Ca2+], in cerebellar granule cells, which can be prevented by inhibitors of NMDA receptors, such as MK-801, suggesting that NO and ONOO~ causes the release of glutamate (16), which then activates its receptors postsynaptically. In these studies, Leist et al. (16) showed that both the intracellular Ca2+ increase and apoptosis elicited by ONOO~ or the NO donors were prevented by blocking exocytosis with tetanus toxin or botulinum neurotoxin C.
These studies suggest that NO toxicity may also be based on its presynaptic actions. In fact, it is known that NO stimulates neurotransmitter release (17,34,35,89-91). Furthermore, Meffert et al. (18) produced evidence showing an increase in docking/fusion of synaptic vesicles in synaptosomes by NO donors, which was abolished by the Clostridium neurotoxins. This would imply that, to some extent, similar mechanisms underly the physiological and the toxic action of NO in neurons in that NO would affect neurotransmitter release by controlling S-nitrosylation of fusion proteins, and whether normal cell communi cation or toxicity occurs would depend on the intensity of the insult (16). Actually, this is similar to what occurs in ischemia and other pathological conditions in which unbalanced release of glutamate contributes significantly to toxicity, since antagonists of the NMDA receptors are neuroprotective (43,45,92,93).
Although ONOO" has been considered a terminal mediator of toxicity, compared to the less reactive NO, these studies indicate that direct exposure of neurons to ONOO" does not result in immediate lethal events and that ONOO", under some conditions, may act by an indirect but specific mechanism in which it stimulates the exocytotic release of glutamate. Thus, ONOO" is capable of recruiting a physiological pathway to initiate damage of neurons exposed to increasing concentrations of NO, and the threshold between fine modulation of neuronal responses in which NO would have a physiological function in stimulating neurosecretion (18) and that for neuronal injury may be decided by the relative concentrations of NO and ONOO.
Pathological situations may bring about inbalances in NO production and in the release and uptake of glutamate, which would cause neurodegeneration. In this respect, NO has been shown to inhibit [3H]glutamate transport in striatal synaptosomes (94), and other studies showed that NO donors (nitroprusside, SNP; S-nitrosoacetylpenicillamine, SNAP) inhibit [3H]glutamate uptake by hip-pocampal synaptosomes from rat brain due to changes in both Km and Vmax of the uptake. The effect of NO donors on glutamate uptake could be reduced by hemoglobin, which binds NO, suggesting that the effects are due to NO (95) or to products of NO metabolism. In fact, studies carried out using reconstituted glutamate transporters revealed that ONOO" inhibits the Vmax of the transport, whereas NO donors failed to significantly modify reconstituted glutamate uptake (96). Therefore, the effects of NO and of NO donors on glutamate uptake in synaptosomes (94,95) are likely to be due to ONOO" formed in the reaction of NO with superoxide. The recombinant rat brain glutamate transporters GLT1, GLAST, and EAAC1 are equally sensitive to ONOO" (96). The observed inhibition by NO/ONOO" of glutamate reuptake may represent a novel type of trans-synaptic retrograde regulation of glutamate transport, involved in the toxic effects of NO and of glutamate, and in more physiological functions, such as long-term potentiation.
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