Role of K(+) efflux in apoptosis induced by AMPA and kainate in mouse cortical neurons.

Activation of ionotropic glutamate receptors can induce neuronal apoptosis in vitro and in vivo. We showed previously that activation of the N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptors in a low Ca(2+) and low Na(+) condition induced apoptotic neuronal death, and that the K(+) efflux via NMDA receptor channels was likely a key event in NMDA-induced apoptosis. Since non-NMDA receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and kainate receptors, are also permeable to K(+), we tested the hypothesis that stimulating K(+) efflux via non-NMDA receptor channels could induce apoptosis in cultured cortical neurons. Using a Ca(2+)-free and Na(+)-free external solution, application of kainate revealed outward membrane currents carried by K(+) efflux. In a low Ca(2+)/low Na(+) medium, a 5-h exposure to 50-500 microM AMPA in the presence of the NMDA receptor antagonist MK801 induced dose-dependent neuronal death 24 h after the onset of the insult, accompanied by intracellular K(+) reduction and caspase-3 activation. The AMPA-induced cell death was attenuated by the caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) and by the protein synthesis inhibitor cycloheximide. Reducing K(+) efflux by raising extracellular K(+) concentration from 5 to 25 mM attenuated AMPA-triggered cell death, the Ca(2+) channel antagonist nifedipine showed no effect on the AMPA toxicity. Kainate induced similar neuronal death sensitive to attenuation by Z-VAD-FMK or elevated extracellular K(+).We suggest that the non-NMDA receptor-mediated K(+) efflux may participate in apoptotic process and that blocking excessive K(+) efflux mediated by NMDA and non-NMDA receptors may selectively prevent neuronal apoptosis under certain pathological conditions.

The industrial chemical Tinuvin 123 does not induce dopaminergic neurotoxicity in C57Bl/6 mice.

We have investigated the acute effects of systemic administration of Tinuvin 123 on nigro-striatal dopaminergic neurons in the C57Bl/6 mouse. Tinuvin 123 was administered subcutaneously (s.c.) twice, 16 h apart, at doses of 0, 2, 20 or 200 mg/kg body weight to a total of 48 male C57Bl/6 mice (12 animals/group). Seven days following the last dose the animals were decapitated and the brains removed. No deaths occurred during the study. There were no differences between the mean body weights of any of the experimental groups prior to or following Tinuvin 123 treatment. Animals treated s.c. with 2 mg/kg Tinuvin 123 exhibited no changes in striatal dopamine or metabolite concentrations compared with vehicle-treated animals. Higher doses of Tinuvin 123 (20 and 200 mg/kg) resulted in a moderate loss of striatal dopamine (31 and 38%) but concentrations of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid and the neurotransmitters serotonin, aspartate, gamma aminobutyric acid and glutamate were unchanged. The total number of tyrosine hydroxylase-immunoreactive neurons in the entire substantia nigra were equivalent in the vehicle- and Tinuvin 123-treated animals at all doses, thus no neuronal loss was demonstrated. In conclusion, this study demonstrates no evidence that systemic administered Tinuvin 123 induces dopaminergic neurotoxicity in C57Bl/6 mice.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline increases extracellular serotonin and stimulates hydroxyl radical production in rats.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a neurotoxin structurally similar to the dopaminergic neurotoxin MPTP, may be formed in humans treated with chloral hydrate or exposed to trichloroethylene, a widely used industrial solvent. Systemically administered TaClo (0.4 mg/kg, i.p.) induced an immediate and transient release of dopamine (DA) and serotonin (5-HT) measured using microdialysis. However, only 5-HT was increased significantly (area under the curve, AUC, for the 1-2 h-period following TaClo administration: 400% compared with the respective control value; 2-3 h-period: 326%). This was followed by a progressive increase in hydroxyl radical formation reflected by higher extracellular concentrations of the hydroxylate product of salicylic acid, 2,3-dihydroxybenzoic acid (AUC for the 1-2 h period following TaClo administration: 182% compared with the respective control value; 2-3 h period: 190%). In contrast, extracellular glutamate and GABA were increased 2-3 h post-injection by 64 and 51%, respectively. These data suggest that TaClo stimulates the generation of hydroxyl free radicals via an acute release of 5-HT and perhaps DA.

Effects of tetraethylammonium analogs on apoptosis and membrane currents in cultured cortical neurons.

Tetraethylammonium (TEA), the quaternary ammonium ion and nonselective K(+) channel blocker, is protective against neuronal apoptosis. We now tested two TEA analogs, tetrapentylammonium (TPeA) and tetrahexylammonium (THA), for their effects on apoptotic neuronal death and for their pharmacological profiles on membrane currents in cultured mouse cortical neurons. TPeA and THA (0.1-1.0 microM) attenuated staurosporine-induced caspase-3 activation and neuronal apoptosis. TPeA and THA blocked the outward delayed rectifier K(+) (I(K)) current in concentration-dependent manners with IC(50) values of 2.7 and 1.9 microM, respectively. I(K) was blocked by TPeA in a use-dependent manner, whereas THA blocked I(K) regardless of activation state of the channel. TPeA at 1 microM inhibited the high voltage-activated (HVA) Ca(2+) current and the A-type K(+) current (I(A)). TPeA (1-10 microM) also blocked the fast inactivating Na(+) current. The ligand-gated N-methyl-D-aspartate (NMDA) receptor current was not affected by up to 20 microM TPeA. THA at 1 microM showed inhibitory effects on I(A), HVA Ca(2+), and Na(+) currents. THA (10 microM) suppressed NMDA currents. The data suggest that, as K(+) channel blockers and apoptosis antagonists, TPeA and THA are much more potent than TEA; however, they have nonspecific actions on several voltage-gated or ligand-gated channels.

The central catechol-O-methyltransferase inhibitor tolcapone increases striatal hydroxyl radical production in L-DOPA/carbidopa treated rats.

Inhibition of catechol catechol-O-methyltransferase (COMT) in the brains of subjects treated with L-DOPA (L-3,4-dihydroxylphenylalanine) and an aromatic amino acid decarboxylase (AADC) inhibitor is suggested to cause an increase of L-DOPA, which might lead to oxidative damage through enhanced formation of free radicals. To investigate this hypothesis, the acute effects of two doses of the systemically administered COMT inhibitors entacapone (peripheral) and tolcapone (peripheral and central) on the extracellular formation of hydroxyl radicals in vivo following treatment with L-DOPA and the AADC inhibitor carbidopa were examined. The formation of extracellular hydroxyl radicals were determined by the measurement of 2,3-dihydroxybenzoic acid (2,3-DHBA), a reaction product of hydroxyl radicals with sodium salicylate, using microdialysis in the striatum of anesthetised rats. The COMT inhibitors were administered together with 50 mg/kg i.p. carbidopa as 5% gum arabic suspensions intraperitoneally (i.p.) at doses of 0, 1.0, and 10 mg/kg body weight to a total of 36 male HAN-Wistars rats. L-DOPA was injected i.p. 40 min after drugs of interest. Microdialysis samples were collected every 20 min for 400 min at a perfusion rate of 1 microl/min. Systemically administered 10 mg/kg tolcapone, but not entacapone, induced an increase in hydroxyl radical formation in the striatum of anesthetised rats following treatment with L-DOPA/carbidopa. The increase in hydroxyl radical formation was reflected by higher extracellular concentrations of the hydroxylate product of salicylate, 2,3-DHBA, peaking at 192% of baseline at the end of the observation period. Similar results were also found using the AUC (area under the curve) value estimated for the observation period. We conclude that the increase in hydroxyl radical formation is likely to result from an increased rate of monoamine oxidase-mediated and non-enzymatic (autoxidation) dopamine metabolism following increased central availability caused by reduction in COMT-mediated metabolism. We cannot, however, exclude the possibility that hydroxyl radicals are produced by tolcapone as a result of uncoupling mitochondrial oxidative phosphorylation.