1.14.1. Astrocytic GSH release
Extracellular GSH has been reported in the micromolar range in the brains of rats (Han et al., 1999). Several studies have reported that cultured rat astrocytes
release GSH into extracellular media (Yudkoff et al., 1990; Sagara et al., 1996; Stone et al., 1999), with approximately 10% of intracellular GSH estimated to be released per hour (Sagara et al., 1996; Dringen et al., 1997a). Despite this release, intracellular astrocytic GSH levels remain unchanged. Neurones either release no GSH or very little (Wang & Cynader, 2000). Inhibition of y-GT with acivicin (Stole et al., 1994) increased the extracellular concentration of GSH detected, indicating that released GSH is a substrate for the enzyme (Dringen et al., 1997a). Furthermore, prolonged incubation (10 hours) with acivicin caused a depletion of intracellular GSH levels, suggesting that the metabolism of GSH by y-GT is required to provide precursors {e.g., cysteine, glycine, and glutamate) for de novo
GSH synthesis (Dringen et al., 1997a). Indeed, when uptake of CysGly (a product of GSH degradation by y-GT; reaction 1.13) in astrocytes was inhibited by blocking the peptide transporter PepT2, astrocytes were unable to maintain intracellular GSH levels (Dringen et al., 1998).
The mechanism by which GSH is released by astrocytes is unknown. Sagara et al
(1996) found that the rate of GSH release was dependent on temperature, and was susceptible to partial inhibition when thiols on the outer leaflet of the plasma membrane were oxidised. These results suggest that a protein transporter mediates GSH efflux from astrocytes. The multidrug resistance protein (MRP) family of transporters have been postulated to release GSH (Yamane et al., 1998; Paulusma
et al., 1999). The MRP family were originally identified as playing a role in the drug resistance of cancer cells (Borst et al., 1999). Over expression of MRP 1 and MRP2 in cultured kidney cells have been shown to increase GSH efflux, while homozygous MRP2 knock-out rats did not release GSH into the bile duct and was concomitant with increased intracellular GSH levels (Paulusma et al., 1999). M RPl, but not MRP2, has been shown to be expressed in rat astrocytes and to facilitate GSSG release (Hirrlinger et al., 2001). No studies on MRPl-mediated GSH release from astrocytes have been reported to date.
1.14.2 Cultured astrocytes and neurones differ in their preference of amino acids for GSH synthesis.
Neurones cultured in isolation are considered to contain less GSH compared to astrocytes cultured in isolation (Sagara et al., 1993; Makar et al., 1994; Bolanos et al., 1995; Dringen et al., 1999b). It has been postulated that the availability of cysteine in culture media may limit the GSH content in neurones (Sagara et al.,
1993; Kranich et al., 1996; Dringen et al., 1999a). GSH levels in cultured neurones can be elevated when incubated with cysteine, but not cystine (Sagara et al., 1993; Kranich et al., 1996). Neurones have been shown to be capable of taking up cystine via both sodium-independent {e.g., Xc- transporter) and sodium- dependent {e.g., Xag- transporter) transport systems (Allen et al., 2001; McBean & Flynn, 2001). This would suggest that the reason why neurones cannot utilise cystine for GSH synthesis is not due to a lack of uptake. Incubation of neurones with glycine or glutamine (as a source for glutamate) had no effect on neuronal GSH levels, indicating that these two precursors of GSH are not limiting (Dringen
et al., 1999a). Astrocytes can utilise either cysteine or cystine as precursors for GSH synthesis (Cho & Bannai, 1990; Sagara et al., 1993; Kranich et al., 1996, 1998), with cystine suggested to be the preferred substrate (Kranich et al., 1996, 1998). Once again, neither glutamate nor glycine appears to limit GSH synthesis in astrocytes (Dringen et al., 1997b).
When neurones are cocultured with astrocytes, neuronal GSH levels are approximately doubled, compared to neurones cultured alone (Sagara et al., 1993; Bolanos et al., 1996; Dringen et a l, 1999a). The release of GSH by astrocytes has been postulated to provide precursors for de novo neuronal GSH synthesis (Dringen et al., 1999a; Wang & Cynader, 2000). Wang and Cynader (2000) have proposed that the GSH released by astrocytes reduces the cystine present in the culture media to cysteine, which can then be taken up by neurones and utilised for GSH synthesis (Figure 1.7, route 1). Alternatively, Dringen et al (1999a) have suggested that the GSH released by astrocytes is metabolised to CysGly by y-GT, with the CysGly then being used by neurones as a precursor for GSH synthesis (Figure 1.7, route 2). In support of this, neurones that are incubated with CysGly
astrocyte
CysGly
AP-N
CysGly
GSH
Cys ^
Cys+Gly
neurone
Cys-Cys
Cys
Figure 1.7. The supply o f neuronal GSH precursors by astrocytes
Astrocytes release GSH which can either (!) reduce cystine (Cys-Cys) to cysteine (Cys), or (2) be metabolised by y-glutamyltranspeptidase (y-GT) to cysteinylglycine (CysGly), which can then be taken up by astrocytes to be recycled into GSH, or hydrolysed by aminopeptidase N (AP-N) on neurones, to generate cysteine and glycine (Gly). The cysteine generated by routes 1 and 2 can then be taken up by neurones for de novo GSH synthesis.
elevate their GSH levels (Dringen et al., 1999a). Furthermore, inhibition of astrocytic y-GT by acivicin prevented the elevation of neuronal GSH levels when they were cocultured with astrocytes (Dringen et al., 1999a). Note that CysGly has also been shown to increase GSH levels in astrocytes (Dringen et al., 1997b), and is thought to be taken up by the PepT2 dipeptide transporter (Dringen et al.,
1998). Indeed inhibition of this transporter causes depletion of GSH in astrocytes (Dringen et al., 1998). Neurones have been shown not to express PepT2 (Dringen
et al., 2001). Instead CysGly has been reported to be hydrolysed by the dipeptidase aminopeptidase N (EC 3.4.11.2), which has been localised to the outer leaflet of the neuronal plasma membrane (Dringen et al., 2001). The cysteine and glycine generated by this enzyme is then taken up by the neurones. Indeed, treatment o f neurones with either CysGly or cysteine + glycine elevated GSH levels to a similar extent (Dringen et al., 1999a, 2001). Furthermore, extracellular cysteine levels are elevated 7-fold when neurones are cocultured with astrocytes (Sagara et al., 1993).