Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l-serine-O-sulphate, but not by l-alpha-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20-24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 +/- 9.8% in neuronal cultures (P < 0.001; n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05; n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 +/- 4.9 micrometer, compared to 1.2 +/- 0.3 micrometer in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 micrometer MK-801, 10 micrometer tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm) inhibited the 45calcium uptake stimulated by 30 micrometer N-methyl-d-aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced approximately 25% inhibition of 30 micrometer NMDA-induced current in cortical neurons. Dihydrokainate (1 mm) alone generated a small current (17% of the current produced by 30 micrometer NMDA) that was blocked by 30 micrometer 5,7-dichlorokynurenate and only weakly by 10 micrometer cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.

Cyanide is a mitochondrial poison that adversely affects cellular respiration by inhibiting the reoxidation of cytochrome a 3 by molecular oxygen, thereby obstructing the electron transport chain and oxidative phosphorylation (1). Cytochrome a 3 and cytochrome a form the cytochrome c oxidase complex, which is the terminal enzyme in the electron transport chain. Transferring electrons to oxygen, cytochrome c oxidase is the cellular respiratory component responsible for the critical need for oxygen. Thus, cyanide intoxication in cells is akin to oxygen starvation. In neuronal systems, cyanide treatment has been a widely used model of hypoxia (2-4), particularly in relation to excitotoxic processes. For example, cyanide can raise extracellular glutamate levels (5, 6), increase glutamate-triggered intracellular Ca 2ϩ elevations in neurons (7,8), and potentiate glutamate toxicity (7, 9). Moreover, specific inhibitors of Nmethyl-D-aspartate (NMDA) 1 receptors can inhibit cyanide-induced Ca 2ϩ influx in neurons (7, 10, 11) as well as neurotoxicity (12-14).Interestingly, a direct interaction between cyanide and the NMDA receptor has recently been demonstrated. Cyanide treatment of cultured rat hippocampal or cerebellar neurons potentiated NMDA-induced physiological responses, including single channel activity in excised outside-out membrane patches (15, 16). However, the precise site of action of cyanide at the receptor remained to be elucidated. In the present investigations we have evaluated the possible interaction of KCN on the NMDA receptor redox modulatory sites (17). Via these sites, disulfide-reducing agents such as dithiothreitol (DTT; see Refs. 17 and 18), dihydrolipoic acid (19), or tris(carboxyethyl)phosphine (20) enhance NMDA receptor-mediated physiological responses, whereas thiol-containing oxidants (21, 22), reactive quinones (23, 24), or oxygen-derived free radicals (25,26) can reverse the effects of reductants or depress native responses. Cyanide has well established properties as a disulfide reducing agent in many preparations (27)(28)(29)(30), and thus an effect on the NMDA thiol-sensitive sites would not be surprising. Nonetheless, the experiments described here demonstrate that cyanide can be used to distinguish between different NMDA receptor subtypes by producing either a potentiation or a depression of the physiological responses mediated by this ligand-gated ion channel. EXPERIMENTAL PROCEDURESTissue Culture-Tissue culture and all common reagents were purchased from Sigma, excluding the following: iron-supplemented bovine calf serum, HyClone Laboratories (Logan, UT), and minimal essential medium, Life Technologies, Inc. Chinese hamster ovary cells (CHO-K1; ATTC CCL61) were grown in Ham's F-12 nutrient medium with 10% fetal bovine serum, and 1 mM glutamine (CHO media) in 50-or 200-ml flasks at 37°C in 5% CO 2 . Cells were passaged at a 1:10 dilution at 80% confluency, approximately every 2 days, no more than 30 times. Cerebral cortices were obtained from E-16 Sprague-Dawley C-D rats and dissocia...

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1 The inhibitory eects of nitric oxide (NO) on N-methyl-D-aspartate (NMDA) receptor function have been proposed to be mediated via the interaction of this gas with a redox-sensitive thiol moiety on the receptor. Here, we evaluated this suggested mechanism by examining the actions of various NO donors on native neuronal receptors as well as in wild-type and cysteine-mutated recombinant NMDA receptors expressed in Chinese hamster ovary (CHO) cells. 2 The NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydraxino)ethanamine (NOC-12; 100 mM) produced a rapid and readily reversible inhibition of whole-cell currents induced by NMDA (30 mM) in cultured cortical neurons. The inhibition was apparent at all holding potentials, though a more pronounced block was observed at negative voltages. The eects of NOC-12 disappeared when the donor was allowed to expire. A similar receptor block was observed with another NO-releasing agent, S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). 3 The blocking eects of NO released by SNAP, 3-morpholinosydnonimine (SIN-1; 1 mM), and 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5; 100 mM) on currents mediated by recombinant NR1/NR2B receptors were virtually indistinguishable from those observed on native receptors. Furthermore, mutating cysteines 744 and 798 of NR1, which constitute the principal redox modulatory site of the NR1/NR2B receptor con®guration, did not aect the inhibition produced by NO. 4 The NR2A subunit may contribute its own redox-sensitive site. However, the eects of NO on NR1/NR2A receptors were very similar to those seen for all other receptor con®gurations evaluated. Hence, we conclude that NO does not exert its inhibition of NMDA-induced responses via a modi®cation of any of the previously described redox-sensitive sites on the receptor.