Excitatory Amino Acids

EAAs, such as glutamate and aspartate, are found in high concentrations in neuronal cells of the central nervous system. EAA receptors are divided into metabotropic and ionotropic receptors. Ionotropic receptors play an important role in neuronal excitotoxicity and include NMDA, AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), and kainate subtypes. Metabotropic receptors are G-protein-coupled and include a family of receptor subtypes, of which mGlul and mGlu2 might play differential roles in ischemic pathogenesis (11). Following energy depletion and anoxic depolarization, massive extracellular glutamate accumulation occurs through presynaptic release and reversal of glutamate transporters in astrocytes (12,13 ). Once released, glutamate binds to its ionotropic receptors, calcium and sodium enter the cell, and a consequent cascade of damaging events ultimately causes cell death.

The NMDA receptor-channel complex is thought to facilitate calcium entry into the cell after stimulation by glutamate or NMDA. At rest, the NMDA channel is blocked by magnesium, which can be removed by a depolarizing stimulus. In addition to its ligand-binding site, the complex contains glycine, polyamine, and zinc domains, of which a variety of modulators have been studied in the laboratory as potential neuroprotectants (13). Unfortunately, none of the therapeutic strategies aimed at these targets have yielded positive results at the clinical level for treatment of acute stroke (14). Other work in this area suggests that modulation of NMDA subunits during ischemia and related insults might have an important role in their function. NMDA receptors consist of several subunits, including NR1 and NR2. NR1 might facilitate neurotoxicity, as NR1 knockout mice were resistant to glutamate-induced excitotoxicity (15) and rats treated with antisense oligodeoxynucleotides to inhibit the synthesis of NR1 receptors had smaller infarcts following experimental stroke (16). Tyrosine phosphorylation of NR2 has been documented following cerebral ischemia (17,18) and has been implicated in the phenomenon of ischemic tolerance, in which a prior sublethal ischemic insult protects against a subsequent lethal insult (19 ) . NMDA receptors interact with a diversity of intracellular

Figure 2 The excitotoxic cascade. In the setting of reduced CBF caused by focal ischemia, energy supplies are depleted, causing membrane depolarization and loss of ionic gradients, which leads to the release of excess excitatory amino acids, such as glutamate. Glutamate activates its ionotropic receptors, permitting entry of large amounts of Ca2+. Excess intracellular Ca2+ then activates a series of events that ultimately lead to cell death. Calcium might activate calpain, leading to degradation of several structural proteins. NMDA-receptor stimulation is linked to the activation of NOS, which generates NO. Calcium also triggers various transcription factors that lead to upregulation of many genes. Some of the upregulated genes are involved in apoptosis and inflammation. Inflammatory responses can lead to increases in ROS, which can lead to further transcriptional responses. Calcium can also generate xanthine oxidase, which, in turn, increases intracellular levels of superoxide. Intracellular Ca2+ also upregulates lipolysis, which, can trigger inflammation through activation of PLA2 and the Arach A cascade. Abbreviations: CBF, cerebral blood flow; NOS, nitric oxide synthase; ROS, reactive oxygen species; NO, nitric oxide; PLA2, phospholipase A2; PKC, protein kinase C; Arach A, arachidonic acid.

Figure 2 The excitotoxic cascade. In the setting of reduced CBF caused by focal ischemia, energy supplies are depleted, causing membrane depolarization and loss of ionic gradients, which leads to the release of excess excitatory amino acids, such as glutamate. Glutamate activates its ionotropic receptors, permitting entry of large amounts of Ca2+. Excess intracellular Ca2+ then activates a series of events that ultimately lead to cell death. Calcium might activate calpain, leading to degradation of several structural proteins. NMDA-receptor stimulation is linked to the activation of NOS, which generates NO. Calcium also triggers various transcription factors that lead to upregulation of many genes. Some of the upregulated genes are involved in apoptosis and inflammation. Inflammatory responses can lead to increases in ROS, which can lead to further transcriptional responses. Calcium can also generate xanthine oxidase, which, in turn, increases intracellular levels of superoxide. Intracellular Ca2+ also upregulates lipolysis, which, can trigger inflammation through activation of PLA2 and the Arach A cascade. Abbreviations: CBF, cerebral blood flow; NOS, nitric oxide synthase; ROS, reactive oxygen species; NO, nitric oxide; PLA2, phospholipase A2; PKC, protein kinase C; Arach A, arachidonic acid.

synaptic and cytoskeletal proteins, and disruption of downstream-signaling pathways also might lead to neuroprotection independent of intracellular calcium changes. NMDA receptor stimulation has been shown to lead to the recruitment of the scaffolding protein postsynaptic density 95 (PSD95), which couples the NMDA receptors to neuronal nitric oxide (NO) synthase (nNOS) (20). nNOS catalyzes the conversion of L-arginine to NO. Prevention of NMDA-PSD95 interactions has led to reduced infarct size and improved neurologic function, without disruption of synaptic transmission or calcium influx. nNOS is now believed to potentiate neuronal cell death in certain settings, as male mice deficient in nNOS have smaller infarcts compared to wildtype mice (21). However, this effect might be gender dependent, as nNOS in female mice might be neuroprotective (22 ).

AMPA receptors are generally permeable to sodium and impermeable to calcium. However, 8% to 15% of brain neurons express calcium-permeable AMPA receptors due to RNA editing of the GluR2 subunit (23). The presence of the GluR2 subunit appears to render AMPA channels calcium impermeable, and cerebral ischemia appears to downregulate GluR2 expression. Thus, excitotoxicity due to AMPA receptor stimulation might also result from increasing Ca2+ influx. Antisense knockdown of GluR2 led to increased calcium influx and increased cell death, even in the absence of ischemia (24). However, the precise role of GluR2 in mediating ischemic injury is not clear. Other studies showed that, although ischemia did reduce GluR2 expression in hippocampal CA1 and CA3 neurons, subsequent neuronal death did not occur (25). Furthermore, mice lacking a functional GluR2 gene did not exhibit increased neurotoxicity, even in the face of increased calcium influx (23). Regardless, AMPA antagonists appear to limit ischemic neuronal damage, especially following global cerebral ischemia (26).

In contrast to the ionotropic receptors, metabotropic receptors are G-coupled proteins with pleitropic effects. The Group 1 mGluRs, including mGluRl and mGluR5, trigger internal calcium release via phosphoinositol and phospholipase C. mGluRl, in particular, appears to play a damaging role following ischemia, and antagonists are neuroprotective in animal models (11).

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