Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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 μm 3H-clorgyline; lane 2, 5 μm 3H-clorgyline; lane 3, 100 μm 3H-clorgyline plus 100 μm deprenyl; and lane 4, 100 μm 3H-clorgyline plus 10 mmunlabeled clorgyline. Molecular weights (in kilodaltons) are indicated at left. Similar results were obtained in two independent experiments.
Tables
Reagent % Survival Control 100 Glutamate (5 mm) 3 ± 1 Kainate (5 mm) 95 ± 5 Aspartate (10 mm) 100 ± 2 AMPA (5 mm) 98 ± 4 ACPD (100 μm) 102 ± 3 NMDA (10 mm) 96 ± 5 NMDA (10 mm) + glycine (5 mm) 97 ± 6 Quisqualate (0.5 mm) 6.1 ± 0.9 Glutamate (5 mm) + APV (10 mm) 1 Glutamate (5 mm) + MK-801 (20 μm) 0 Cystine (1 mm) 100 ± 5 Glutamate (5 mm) + cystine (1 mm) 95 ± 8 -
HT-22 cells were incubated with the above reagents for 24 hr, and then cell viability was assessed using the MTT assay. Data are expressed as percent survival relative to untreated controls and are the means of triplicate determinations ± SD. The results were confirmed by visual inspection of the cells. APV, 2-amino-5-phosphonovalerate; ACPD, 1-aminocyclopentane-1,3 dicarboxylic acid. ACPD is a selective metabotropic glutamate receptor agonist.
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Treatment % MAO activity Clorgyline (1 μm) 12.8 ± 4.3 Clorgyline (10 μm) 15.2 ± 2.5 Clorgyline (100 μm) 18.9 ± 2.8 Pargyline (1 μm) 46.5 ± 15.3 Pargyline (10 μm) 19.7 ± 2.9 Pargyline (100 μm) 17.6 ± 2.5 Harmine (1 μm) 10.3 ± 4.2 Harmine (10 μm) 10.9 ± 4.9 Harmine (100 μm) 18.9 ± 6.0 Hydralazine (1 μm) 74.9 ± 3.2 Hydralazine (10 μm) 40.8 ± 7.1 Hydralazine (100 μm) 13.6 ± 8.2 DPI (1 μm) 92.2 ± 4.6 -
The activity of MAO in intact HT-22 cells was determined by using a slight modification of the method of Edelstein and Breakefield (1981). The results are presented as the percentage of the value obtained with the untreated control (300–400 pmol · min−1 · mg−1 protein) ± SD. Similar results were obtained in two to three separate experiments.
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- Table 3.
Effect of monoamine uptake inhibitors on MAO activity in whole cells versus cell homogenates
Treatment % MAO activity Intact cells Imipramine (10 μm) 89.4 ± 18.0 Imipramine (75 μm) 32.9 ± 11.0 Doxepin (10 μm) 91.9 ± 10.0 Doxepin (75 μm) 42.5 ± 3.6 Clomipramine (10 μm) 82.7 ± 10.0 Clomipramine (30 μm) 41.5 ± 15.0 Cell extracts Imipramine (10 μm) 95.4 ± 3.2 Imipramine (75 μm) 75.0 ± 0.1 Doxepin (10 μm) 96.8 ± 2.3 Doxepin (75 μm) 77.5 ± 2.7 Clomipramine (10 μm) 90.8 ± 5.0 Clomipramine (30 μm) 79.0 ± 4.0 -
The activity of MAO in intact HT-22 cells was determined by using a slight modification of the method of Edelstein and Breakefield (1981). The activity of MAO in cell extracts was determined by the method of Wurtman and Axelrod (1963). The results are presented as the percentage of the value obtained with the untreated control ± SD. Similar results were obtained in two to three separate experiments.
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Treatment − Glutamate + Glutamate Control 100 ± 17 139 ± 23* Clorgyline (100 μm) 76 ± 12 77 ± 12 Deprenyl (100 μm) 120 ± 18 149 ± 23* Imipramine (75 μm) 142 ± 15 135 ± 16 DPI (1 μm) 192 ± 31 210 ± 31 Quinacrine (10 μm) 151 ± 12 157 ± 12 -
HT-22 cells were incubated for 6–8 hr in the presence or absence of 5 mm glutamate with the MAO inhibitors clorgyline and deprenyl, the monoamine uptake inhibitor imipramine, or the NADPH oxidase inhibitors diphenylene iodinium (DPI) and quinacrine. The levels of intracellular peroxides were estimated by FACS analysis as described in Materials and Methods. Because quantitative measurements are subject to error attributable to the nonratiometric emission of the dye, data are presented as a percentage change relative to control cells arbitrarily set at 100%. Forward scatter measurements determined that cell volume remained constant across the experimental procedures. The data are expressed as the mean ± SD of 10,000 independent measurements. With the exception of the points marked with an asterisk, all values for glutamate-treated cells were not significantly different from those for untreated cells. *p < 0.005. Repeated three times with similar results.
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