BRAIN RESEARCH ELSEVIER Brain Research 716 (1996) I IX-122 Research report Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage Jane E. Barker “I. Simon J.R. Heales a.b,Adrian Cassidy “, Juan P. Bolaiios ‘, John M. Land “h, John B. Clark a Accepted 20 December 1995 Abstract Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson’s disease (PD). While the cause of the loss is unclear. an imbalance in the enzymes associated with the synthesis, utilisation. degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: il regenerates GSH from the oxidised form (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In vie* of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats. brain GSH was depleted by up to 60% by subcutaneous administration of I.-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro. using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurrin, 0 at an initial concentration of 0.09 mM. These data suggest that GSH is Important in the maintenance glutathione reductase actlvlty. This may arise in part from its ability to inactivate oxidising agents such as ONOO-. 1. Introduction The pathological characteristic of Parkinson’s disease (PD) is the death of dopaminergic neurones in the substan- tia nigra (SN) [5]. While the cause of the neurodegenera- tion is undefined, several processes have been implicated including alterations in the protective mechanisms against neurotoxic oxidising species [9]. A major intracellular antioxidant of the mammalian brain is glutathione (GSH); together with glutathione peroxidase it is the major protec- tion against hydrogen peroxide accumulation and against derived oxygen species [d]. In vitro studies have correlated loss of cellular GSH with susceptibility of neurones to the oxidant peroxynitrite [2]. An important clinical corollary to the observation is the early and selective loss of GSH from the SN of PD patients [9]. Corresponding author. Fax: (44) (7 I) X33- ltJl6. 0006-8993/9hjS I5.00 0 I996 Elsevizt- Science B.V PI/ SOOO6-8993~96~00003-0 While the initial cause of depletion of GSH in PD is unknown, several processes have been implicated includ- ing an imbalance in GSH homeostasis. The level of GSH is maintained by a number of enzymes: y-glutamyl cys- teine synthetase, the rate limiting enzyme for GSH biosyn- thesis; glutathione peroxidase, which in the presence of hydrogen peroxide oxidises GSH to oxidised glutathione (GSSG); glutathione transferase. responsible for the forma- tion of mercapturic acids and y-giutamyl transpeptidase, involved in the degradation and translocation of GSH [ 141. In conditions such as PD, the activities of the above enzymes have been documented [22]. The observation that y-glutamyl transpeptidase activity was selectively in- creased in the SN of PD patients. suggested that disruption of GSH homeostasis was associated with PD [22]. Another mechanism important in the homeostasis of GSH is the recycling of GSH from the oxidised form. GSSG, a process catalysed by the enzyme glutathione reductase. This enzyme. by reduction of GSSG, regener-