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. 2002 Nov 1;22(21):9203-9.
doi: 10.1523/JNEUROSCI.22-21-09203.2002.

Barbiturates induce mitochondrial depolarization and potentiate excitotoxic neuronal death

Christopher M Anderson  1 , Becky A Norquist, Sabino Vesce, David G Nicholls, William H Soine, Shumin Duan, Raymond A Swanson
Affiliations

Barbiturates induce mitochondrial depolarization and potentiate excitotoxic neuronal death

Christopher M Anderson et al. J Neurosci. .

Abstract

Barbiturates are widely used as anesthetics, anticonvulsants, and neuroprotective agents. However, barbiturates may also inhibit mitochondrial respiration, and mitochondrial inhibitors are known to potentiate NMDA receptor-mediated neurotoxicity. Here we used rat cortical cultures to examine the effect of barbiturates on neuronal mitochondria and responses to NMDA receptor stimulation. The barbiturates tested, secobarbital, amobarbital, and thiamylal, each potentiated NMDA-induced neuron death at barbiturate concentrations relevant to clinical and experimental use (100-300 microm). By using rhodamine-123 under quenching conditions, barbiturates in this concentration range were shown to depolarize neuronal mitochondria and greatly amplify NMDA-induced mitochondrial depolarization. Barbiturate-induced mitochondrial depolarization was increased by the ATP synthase inhibitor oligomycin, indicating that barbiturates act by inhibiting electron transport sufficiently to cause ATP synthase reversal. Barbiturates similarly amplified the effects of NMDA on cytoplasmic free calcium concentrations. The cell-impermeant barbiturate N-glucoside amobarbital did not influence mitochondrial potential or potentiate NMDA neurotoxicity or calcium responses. However, all of the barbiturates attenuated NMDA-induced calcium elevations and cell death when present at millimolar concentrations. Whole-cell patch-clamp studies showed that these effects may be attributable to actions at the cell membrane, resulting in a block of NMDA-induced current flux at millimolar barbiturate concentrations. Together, these findings reconcile previous reports of opposing effects on barbiturates on NMDA neurotoxicity and show that barbiturate effects on neuronal mitochondria can be functionally significant. Effects of barbiturates on neuronal mitochondria should be considered in experimental and clinical application of these drugs.

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Figures

Fig. 1.
Fig. 1.
Effects of barbiturates on NMDA neurotoxicity.A, Exposure to NMDA for 5 min resulted in neuronal death with an EC50 of ∼200 μm. B, Secobarbital at 100 and 300 μm potentiated NMDA neurotoxicity, but this effect was reversed at higher secobarbital concentrations. Vertical hatches mark the beginning of the log scale. ∗∗p < 0.01 versus the 0 secobarbital control in each of the two treatment groups;n ≥ 7. C, The thiobarbiturate thiamylal produced a similar biphasic effect. For both thiamylal and secobarbital, MK-801 completely blocked the toxicity produced by NMDA plus barbiturate. ∗∗p < 0.01 versus the 0 barbiturate group; ††p < 0.01 between indicated groups; n ≥ 7. All data are means ± SE. C, Data are shown after subtraction of 12% neuronal death observed in the control (wash only) condition.
Fig. 2.
Fig. 2.
Comparisons between a cell-permeant and a cell-impermeant barbiturate. A, The cell-impermeant barbiturate N-glucoside (N-glu) amobarbital reduced NMDA toxicity at all concentrations tested, whereas the parent compound had a biphasic effect on NMDA toxicity. Vertical hatches mark the beginning of the log scale. B, Representative patch-clamp recordings from different neurons exposed to NMDA in the presence of sequentially elevated concentrations of secobarbital orN-glucoside amobarbital. C, Pooled data showing a concentration-dependent inhibition of the NMDA current with both barbiturates. Currents from each recorded neuron are normalized to the peak current recorded in the absence of barbiturate. Data are means ± SE; ∗p < 0.05; ∗∗p < 0.01 versus control;n = 3–6.
Fig. 3.
Fig. 3.
Barbiturates cause mitochondrial depolarizationin situ. A, Secobarbital induced an increase in total cell rhodamine-123 fluorescence, indicative of mitochondrial depolarization. The addition of oligomycin caused additional depolarization, indicating that secobarbital induces ATP synthase reversal. B, The cell-impermeable barbiturateN-glucoside amobarbital had no effect on ΔΨm. Oligomycin caused a slight membrane hyperpolarization. C, Secobarbital and the complex 1 inhibitor rotenone increased peak mitochondrial depolarization to a plateau at approximately −110 mV, whereas the uncoupler FCCP increased peak depolarization dose dependently. The EC50 for secobarbital is ∼30 μm. Data in A andB are representative traces. Data inC are means ± SE of ≥10 neurons from two different coverslips.
Fig. 4.
Fig. 4.
Secobarbital potentiates NMDA-induced mitochondrial depolarization. A, Bath-applied NMDA (40 μm) produced an increase in total cell rhodamine-123 fluorescence, indicative of a slight mitochondrial depolarization.B, Pretreatment with 100 μm secobarbital enhanced NMDA-induced depolarization. Data are representativetraces of 12 individual neurons from two separate coverslips for each treatment regimen.
Fig. 5.
Fig. 5.
Barbiturate effects on NMDA-induced cytoplasmic Ca2+ elevations. A, A 5 μm concentration of NMDA alone produced a small rise in intracellular Ca2+. This increase was significantly increased in the presence of 100 μm secobarbital but nearly eliminated in the presence of 1 mm secobarbital.Secobarb, Secobarbital. B, The larger increase in intracellular Ca2+ induced by 20 μm NMDA was reduced by both 1 mm amobarbital and 1 mmN-glucoside amobarbital. Error bars are omitted for clarity. Values adjacent to the tracings are the mean ± SE of the integrated NMDA-induced changes in the fura-2 fluorescence ratio integrated over the 4 min interval beginning 10 sec after the addition of NMDA. ∗∗p < 0.01 versus NMDA alone; n = 35–49 in A;n = 12–19 in B. Barb, Barbiturate.
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References

    1. Airey IL, Smith PA, Stoddart JC. Plasma and cerebrospinal fluid barbiturate levels during prolonged continuous thiopentone infusion. Anaesthesia. 1982;37:328–331. - PubMed
    1. Aldrich WN, Parker VH. Barbiturates and oxidative phosphorylation. Biochem J. 1960;76:47–56. - PMC - PubMed
    1. Barker JL, Ransom BR. Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture. J Physiol (Lond) 1978;280:355–372. - PMC - PubMed
    1. Beal MF. Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann Neurol. 1992;31:119–130. - PubMed
    1. Bhayana V, Alto LE, Dhalla NS. Effects of pentobarbital and pentothal on rat heart contractile force and oxidative phosphorylation activities. Gen Pharmacol. 1980;11:375–377. - PubMed

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