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. 1996 Oct 15;16(20):6394-401.
doi: 10.1523/JNEUROSCI.16-20-06394.1996.

The role of monoamine metabolism in oxidative glutamate toxicity

P Maher  1 , J B Davis
Affiliations

The role of monoamine metabolism in oxidative glutamate toxicity

P Maher et al. J Neurosci. .

Abstract

Glutamate kills neuronal cells by either a receptor-mediated pathway or the inhibition of cystine uptake, the "oxidative pathway." Antioxidants can block cell death initiated by either pathway, suggesting that toxicity is dependent on the production of free radicals. We provide evidence that in a neuronal cell line, glutamate toxicity via the oxidative pathway requires monoamine metabolism as a source of free radicals. Glutamate toxicity is inhibited by monoamine oxidase (MAO) type-A-specific inhibitors, but only at concentrations much higher than those required to inhibit classical type-A MAO. Toxicity is not inhibited by MAO type-B-specific inhibitors at any concentration. Furthermore, treatment of cells with agents that block monoamine uptake inhibits glutamate toxicity. These results suggest that an enzyme distinct from MAO is involved in monoamine metabolism and demonstrate a relationship between glutamate toxicity and monoamine metabolism. These data also have implications for the understanding and treatment of neurodegenerative disorders in which glutamate toxicity is thought to be involved.

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Figures

Fig. 1.
Fig. 1.
Rescue of neuronal cells from glutamate toxicity by MAO inhibitors. A, HT-22 cells were incubated with 100 μm deprenyl, 100 μm pargyline, 100 μm RO16-6491, 100 μm TC715, 100 μm hydralazine, 100 μm clorgyline, 50 μm harmine, 100 μm RO41-1049, or 100 μm TC724 for 8 hr in the presence of 5 mmglutamate. After 24 hr, cell viability was assessed using the MTT assay. Data are expressed as % survival relative to controls treated with inhibitor alone and are the mean of triplicate determinations ± SD. Statistical analysis of the results showed that the MAO-A inhibitors provided significant protection (p < 0.0001) from glutamate toxicity, whereas the MAO-B inhibitors did not. Similar results were obtained in five separate experiments. B, HT-22 cells were exposed to an increasing dose of harmine (▪), clorgyline (•), or deprenyl (○) in the absence or presence of 10 mm glutamate. After 24 hr, cell viability was assessed using the MTT assay. Data are expressed as % survival relative to controls treated with inhibitor alone and are the mean of triplicate determinations ± SD. Similar results were obtained in three separate experiments.
Fig. 2.
Fig. 2.
Primary cortical cells were exposed to 5 mm glutamate (Glu) and clorgyline (Clorg), harmine (Harm), or deprenyl (Dep) at the indicated concentrations. After 24 hr, surviving neurons were counted as described (Murphy et al., 1990). Survival after glutamate exposure is expressed as the % of the mean number of neurons counted in control cultures treated with inhibitor alone. Values represent the mean of triplicate determinations ± SD. Statistical analysis of the results showed that clorgyline and harmine provided significant protection (p < 0.0001) from glutamate toxicity, whereas deprenyl did not. All experiments were repeated at least twice with similar results.
Fig. 3.
Fig. 3.
A, Rescue of neuronal cells from glutamate toxicity by monoamine uptake inhibitors. HT-22 cells were incubated with 75 μm doxepin, 75 μmimipramine, 30 μm clomipramine, 200 μmalaproclate, or 10 μm indatraline for 8 hr in the presence of 5 mm glutamate. % survival was measured after 24 hr by the MTT assay. Data are expressed as % survival relative to controls treated with inhibitor alone. The concentrations of inhibitors used in this experiment afforded maximal protection without causing significant cell death. At higher concentrations the uptake inhibitors were all extremely toxic to the cells. Values represent the mean of quadruplicate determinations ± SD. Statistical analysis of the results showed that the uptake inhibitors provided significant protection (p < 0.0001) from glutamate toxicity. Similar results were obtained in five separate experiments.B, Primary cortical cells were exposed to 5 mm glutamate and 25 μm imipramine, 25 μm doxepin, 10 μm clomipramine, 10 μm indatraline, or 0.1 μm DPI. After 24 hr, surviving neurons were counted as described (Murphy et al., 1990). Survival after glutamate exposure is expressed as the % of the mean number of neurons counted in control cultures treated with inhibitor alone. Values represent the mean of triplicate determinations ± SD. Statistical analysis of the results showed that both the uptake inhibitors and DPI provided significant protection (p < 0.0001) from glutamate toxicity. All experiments were repeated at least twice with similar results.
Fig. 4.
Fig. 4.
Effect of culture medium on glutamate toxicity in neuronal cells. HT-22 cells were incubated for 8 hr plus (hatched bars) or minus (solid bars) 5 mm glutamate in DMEM containing 10% FCS (DME + serum), N2 medium (N2), DMEM containing 10% charcoal-treated serum (charcoal-treated) (Vrana et al., 1993), N2 medium supplemented with 100 μm dopamine (N2 + dopamine), N2 medium supplemented with 100 μm dopamine and 75 μm imipramine (N2/dopamine + imip.), N2 medium supplemented with 100 μm dopamine and 75 μm doxepin (N2/dopamine + dox.), or N2 medium supplemented with 100 μm dopamine and 20 μm indatraline (N2/dopamine + indat.). % survival was measured after 24 hr by the MTT assay, except in the case of the experiments with dopamine, in which survival was measured by the colony-forming assay because dopamine interfered with the MTT assay. Data are expressed as % survival relative to controls treated with the complete medium alone (DME + serum). Values represent the mean of quadruplicate determinations ± SD. Statistical analysis of the results showed that both N2 medium and charcoal-treated serum provided significant protection from glutamate toxicity (p < 0.0001). Dopamine eliminated this protection, but the further addition of the uptake inhibitors provided significant protection (p < 0.001) from the toxicity seen in the presence of glutamate and dopamine. Similar results were obtained in three separate experiments.
Fig. 5.
Fig. 5.
Oxidase inhibitors protect neuronal cells from glutamate toxicity. HT-22 cells were incubated with 1 μmDPI or 10 μm quinacrine for 8 hr in the presence of 5 mm glutamate. % survival was measured after 24 hr by the MTT assay. Data are expressed as % survival relative to controls treated with inhibitor alone. The concentrations of inhibitors used in this experiment afforded maximal protection without causing significant cell death. Values represent the mean of quadruplicate determinations ± SD. Statistical analysis of the results showed that both DPI and quinacrine provided significant protection (p < 0.0001) from glutamate toxicity. Similar results were obtained in five separate experiments.
Fig. 6.
Fig. 6.
3H-clorgyline binding to HT-22 mitochondria. Mitochondria were prepared from low-density cultures of HT-22 cells and labeled with different concentrations of3H-clorgyline in the absence or presence of the indicated antagonists, as described in Materials and Methods. Lane 1, 100 μm3H-clorgyline; lane 2, 5 μm3H-clorgyline; lane 3, 100 μm3H-clorgyline plus 100 μm deprenyl; and lane 4, 100 μm3H-clorgyline plus 10 mmunlabeled clorgyline. Molecular weights (in kilodaltons) are indicated at left. Similar results were obtained in two independent experiments.
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