Sxc- is a particularly intriguing transporter from a functional perspective, because both the import of L-Cys2 and the export of L-Glu are each associated with a distinct set of physiological roles within the CNS. Until only recently, most studies on Sxc- have primarily focused on its significance as a rate-limiting step in the provision of intracellular cysteine needed to maintain appropriate levels of the antioxidant glutathione (GSH). Given the sensitivity of the CNS to oxidative pathology it is not surprising that specific pathways have evolved to ensure neurons and glia have adequate capacities to produce GSH. Although both cell types require intracellular CysH to synthesize GSH, the extracellular precursor and route of entry necessary to provide this CysH appear to differ. Thus, a Cys2/CysH shuttle has been proposed in which the CysH needed to maintain neuronal GSH levels is ultimately dependent upon the Sx--mediated uptake of Cys2 into astrocytes and its subsequent efflux from the cell as either CysH or GSH [122,163-165]. The significance of Sx- in GSH synthesis is underscored by the fact that QA-mediated toxicity in some cells is a consequence of blocking this transporter and the resultant oxidative stress, rather than the result of EAA receptor-mediated excitotoxic-ity [99,149].
Within this protective context, Sx- expression is a part of a number of different adaptive cellular responses (e.g., amino acid starvation, ox-idative stress, toxic exposure) that are under transcriptional control regulated by genomic cis-elements. In the most thoroughly studied cases, exposure to electrophiles and/or increased oxidative stress activates transcription factors (e.g., Nrf2) that bind to electrophilic-responsive elements (EpRE)/antioxidant-responsive elements (ARE) and result in the up-regulation of proteins presumed critical to detoxification and/or antioxidant defense mechanisms, including: GSH transferase, y-glutamylcysteine synthetase, NAD(P)H: quinone reductase, heme-oxygenase 1 and Sx- [166-169]. The transporter is also present (and inducible) at the blood-brain barrier, where it may serve as a point of entry not only for L-Cys2, but also structurally related drugs and neurotoxins (e.g., ^-L-ODAP, see above) [124,151, 164,170]. Surprisingly, the level of Sx--mediated uptake typically reported to be present in primary cultures of astrocytes is remarkably low. This incon-
sistency was largely resolved when it was found that Sx- activity is markedly up-regulated when the neonatal astrocyte cultures are differentiated with dibutyryl-cAMP and adopt a morphology more closely resembling that observed in vivo .
The Cys2/CysH shuttle model, and its dependence upon Sx- is, however, not without controversy. Indeed, it has been reported that the EAATs may provide another route of entry of Cys2 into either neurons or glia [133,171, 172]. Interestingly the EAATs, particularly neuronal EAAT 3, have also been postulated to transport CysH selectively into neurons [173,174]. This role has received considerable support with the finding that EAAT 3/EAAC 1-null mice exhibit markedly lower levels of neuronal GSH, increased markers of oxidative damage, enhanced sensitivity to oxidative stress, and exhibit age-dependent neurodegeneration .
As an obligate exchanger, the import of L-Cys2 is coupled to the export of an equivalent amount of L-Glu. While this allows cells to utilize high intra-cellular concentrations of L-Glu as a driving force for the uptake of needed L-Cys2, it also provides a route for the accumulation of L-Glu in the extracellular space that, if not adequately regulated by the EAATs, could potentially trigger an excitotoxic response. Thus, in the two disorders where the evidence is strongest for Sx- acting as a source for excitotoxic levels of L-Glu, CNS infection and glial tumors, it is not surprising that both of the cell types involved express enriched levels of the transporter. In CNS infection and inflammation, the cellular source of L-Glu are microglia, whose oxidative-based defense mechanisms necessitate high levels of Sxc- activity to provide the Cys2/CysH necessary to maintain an adequate glutathione supply. Ironically, it thus appears that the excitotoxic pathology associated with a number of infections may actually be a secondary result of the migration of high numbers of microglia into an area and a subsequent release of L-Glu as these cells import the L-Cys2 integral to their protective roles [135,175].
Similarly, astrocytoma cells express markedly higher levels of Sx- [153, 160]. Numerous cellular and functional changes accompany the progression from low-grade astrocytomas to high-grade glioblastoma multiforme tumors. Of particular interest is the observation that the increase in Sxc-expression is also accompanied by a reduction in EAAT-mediated transport of glutamate . Several studies utilizing human tumor cell lines and animal models now suggest these tumors employ the mechanism of ex-citotoxicity, in part by the release of excessive glutamate through Sx-, to actively kill surrounding neurons in the peritumoral space to aid tumor expansion [138,160,176]. Epileptic seizures associated with brain tumors are also believed to result from this released glutamate. Further, the concurrent ability to acquire more L-Cys2 and maintain high levels of GSH allows the tumor to survive the necrotic biochemical environment engulfing the tumor. As a transporter, Sxc- may also impact the chemosensitivity and chemoresis-tance of a broad range of tumors through the uptake and efflux of anticancer agents [153,159,177]. The application of identified Sx- inhibitors, (S)-4-CPG or sulfasalazine (Fig. 8j) to a series of glioma cell lines produced a marked reduction in L-Cys2 uptake and intracellular GSH concentration whilst also inhibiting tumor cell growth and inducing caspase-dependent apoptotic cell death. Sulfasalazine was also demonstrated to suppress tumor growth in vivo. In addition to inhibiting the uptake of L-Cys2, L-alanosine has been shown to induce cytotoxicity in lung and ovarian cancer cell lines as a consequence of its intracellular accumulation following uptake through Sx-. Consistent with such a mechanism, inhibition of Sx- with S-4-CPG decreased the cytotoxicity of L-alanosine. In contrast, the cytotoxic potential of geldanamycin was increased following inhibition of Sx-, as the subsequent reduction in GSH level attenuated the detoxification of this drug. This suggests a multidrug strategy based on substrate and/or inhibitor activity may be a relevant therapeutic approach in treating tumors that needs to be further investigated.
Significantly, evidence is beginning to emerge that this export of L-Glu through Sx- may be relevant to more than just pathological mechanisms and may actually represent a novel route of release through which L-Glu can activate extrasynaptic EAA receptors. Based primarily on microdialysis data, work by Kalivas, Baker and coworkers report that Sxc- appears to be a primary source of extracellular L-Glu in select brain regions, such as the striatum and nucleus accumbens [100-102]. Indeed in vivo microdialysis has revealed Sx-is the primary source of non-vesicular extracellular glutamate outside the synaptic cleft and responsible for as much as 50-70% of the basal extracellular level in the nucleus accumbens (NAc). Consistent with this hypothesis, inhibition of Sx- with 4-S-CPG reduced extracellular L-Glu level, as well as blocked the accumulation of L-Glu produced by inhibiting EAAT activity. This efflux of L-Glu is thought to regulate synaptic release (of both L-Glu and dopamine) through the tonic activation of extrasynaptic Group II mGluRs, which have been implicated in various forms of neuroplasticity and neuro-physchiatric disorders. Of particular significance, these extracellular levels of L-Glu are reduced in the nucleus accumbens during cocaine addiction and withdrawal, an effect attributed to the decreased function of Sx- . Consistent with this model, the infusion of Cys2 restored L-Glu levels and, importantly, treatment of rats with the CysH/Cys2 prodrug N-acetyl-CysH prevented reinstatement in cocaine-addicted rats. This led to the conclusion that the increased susceptibility to relapse that accompanies withdrawal from cocaine addition is linked to the decreased activity of Sx-, reductions in extracellular L-Glu levels, and consequent loss of mGluR-mediated regulation of excitatory transmission. Remarkably restoring Sxc- activity, presumably with increased substrate levels, prevents "cocaine-primed drug seeking" [102, 178]. This suggests that in addition to receptor-targeted approaches, agents directed at Sx- may hold therapeutic value in the treatment of addiction.
This postulated role of Sx- (and Cys2) in regulating extracellular L-Glu level is, however, not without its controversies and complications. For ex-
ample, very similar microdialysis studies in prefrontal cortex demonstrated that inhibition of Sxc- did not reduce extracellular L-Glu concentrations, yet did block the accumulation of L-Glu produced following the inhibition of EAAT activity . All of these studies are also complicated by the fact that the Sxc- inhibitor most often employed was 4-S-CPG, which is also an mGluR1/5 antagonist. Ironically, during the course of the studies another mGluR1/5 antagonist (LY367385) was also found to be an Sx- inhibitor . In related studies by Atwell and coworkers, an L-Cys2-mediated efflux of L-Glu through Sxc- was shown to be significant enough to activate non-NMDA in cerebellar slices and NMDA receptors in hippocampal slices [180,181]. However, it was concluded that given both CSF levels of L-Cys2 (typically in the 0.1-0.5 |M range) and its Km values at Sx- (typically reported in 100 |M range), it was unlikely that this mechanism contributed to tonic glutamate levels (and EAA receptor signaling) under normal physiological conditions. This conclusion, however, must be tempered somewhat by direct measurements of 35S-L-Cys2 uptake in slices of nucleus accumbens which yielded Km values in the 2-4 |M range . Further, an Sx--mediated efflux of L-Glu was also shown capable of decreasing the synaptic release of L-Glu (decreased mEPSC and spontaneous EPSC frequency) as a consequence of presynap-tic mGluR2/3 activation by levels of L-Cys2 in the 0.1-0.3 |M range . Lastly, recent studies in which homologues of Sx- (e.g., genderblind) have been genetically eliminated in Drosophila support the participation of Sx- in the regulation of extracellular L-Glu levels and further suggest a role of this extracellular L-Glu in iGluR desensitization and clustering .
Taken together, these findings suggest that Sxc- may indeed function to regulate extracellular L-Glu levels and set a tone at extrasynaptic mGluRs, although this contribution may be a function of the synaptic circuit being examined, the activity of Sxc- and EAATs, as well as extracellular Cys2 levels. A particularly intriguing aspect of these variables concerns the balance set between these two transporters, with respect to kinetic properties, expression levels and localizations within the microenvironment of extrasynaptic receptors. Changes in any one of these properties, whether the result of development, plasticity, or pathology could impact excitatory signaling and/or excitotoxic vulnerability. Assessing the coordinate activity and functions of the EAATs and Sx- will be dependent upon the continued development of potent, selective inhibitors and substrates in combination with more thorough understanding of the structure-activity relationships that govern binding and uptake.
Acknowledgements The authors are grateful to M.P. Kavanaugh, S.E. Esslinger, J.M. Gerdes, N. Natale, C.M. Thompson and P. Kalivas for their insightful discussions and comments. This work was supported in part by NINDS NS30570 and NCCR COBRE RR15583.
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