Peroxynitrite Inhibits Glutamate Transporter Subtypes

Abstract

The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H2O2). Here we show that another biological oxidant, peroxynitrite (ONOO-), formed by combination of superoxide (O2-) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO- reduces selectively the Vmax of transport; its action is fast (reaching > or = 90% within 20 s), dose-dependent (50% inhibition at 50 microM), persistent upon ONOO- (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO- with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO . H2O2 also inhibits reconstituted transport, and its action matches that of ONOO- on all respects; however, this is observed only with 5-10 mM H202 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM), does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO- and other oxidants.

Induction of Cyclooxygenase-2 Accounts for Restraint Stress-Induced Oxidative Status in Rat Brain

“…Although glutamate concentration in this report has been determined in plasma, its increase after stress very likely reflects the profile of this EAA in the brain, as has been shown to be the case after cerebral ischemia (Baker et al, 1995;Castillo et al, 1997;De Cristóbal et al, 2001). It is well known that one of the main mechanisms of glutamate toxicity in neurodegenerative diseases is the oxidation of transporters which account for inhibition of EAA reuptake (Trotti et al, 1996). Although the present study focuses on changes that occurred after an acute unique stress session of 6 h, the fact that specific blockade of NMDA receptors prevented both COX-2 activity and expression could indicate a new neuroprotective mechanism of strategies minimizing excitotoxicity, although caution should be emphasized with the use of pharmacological tools blocking physiological NOS-1 production of NO.…”

Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosis

“…A second homeostatic response of EAATs appears to result from oxidative stress, which exerts deleterious actions on EAATs (Keller et al, 1997;Pedersen et al, 1999;Trotti et al, 1996;Volterra et al, 1994). Both the oxidative and excitotoxic treatments mediated changes in astrocytic phenotype, which were much more prominent following oxidative insult, typical of reactive astrocytes (Pekny and Nilsson, 2005), and likely to improve handling of extracellular Glu (Escartin et al, 2006).…”

“…Oxidative mediators chosen were based on their relevance to ALS: 3-morpholinosydnonimine chloride (SIN-1; Barbeito et al, 2004;Trotti et al, 1996), trans-4-hydroxy-2-nonenal (4-HNE; Blanc et al, 1998;Keller et al, 1997;Pedersen et al, 1998Pedersen et al, , 1999 and hydrogen peroxide (H 2 O 2 ; Gurney et al, 1998). For comparison, an excitotoxic insult with the potent AMPA receptor agonist, (S)-5-fluorowillardiine (FW; Patneau et al, 1992), was chosen based on the AMPA receptor-mediated calcium permeability of motoneurons (Greig et al, 2000;Van Den Bosch et al, 2000), which makes them susceptible to excitotoxicity.…”

“…High levels of oxidative stress result in early reactive changes to astrocytic morphology and redistribution of EAATs. An increase in EAAT affinity also occurs, compensating for RONS-mediated inhibition of EAAT function (Keller et al, 1997;Pedersen et al, 1999;Trotti et al, 1996;Volterra et al, 1994) and preventing increases in extracellular Glu and neurotoxicity. Given the importance of the astrocytic EAATs in maintaining Glu homeostasis (Rothstein et al, 1996), compensatory changes to EAAT activity may reflect an early neuroprotective component of the diverse spectrum of responses of reactive astrocytes (Pekny and Nilsson, 2005;Ridet et al, 1997).…”

Amyotrophic Lateral Sclerosis-linked Glutamate Transporter Mutant Has Impaired Glutamate Clearance Capacity

“…The different transporters of this family share similar structural traits and exhibit different yet comparable functions. Recent studies in several laboratories have provided significant advances in the understanding of the structure and function of these transporters (13,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). These findings constitute a new direction in our knowledge of ion coupling, stoichiometry, membrane protein structure, and pathophysiological implications of transporters.…”