Cyclothiazide binding to the GABAA receptor.

In order to explore the molecular interaction between cyclothiazide (CTZ) and gamma-aminobutyric acidA (GABAA) receptors, possibly underlying inhibition of GABAA receptor currents, [3H]-CTZ was synthesized. Binding of [3H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (-)[1S,9R]-bicuculline methiodide (BMI) (EC(50,BMI)=500+/-80microM). GABA decreased [(3)H]-CTZ binding induced by the presence 300microM and 3mM BMI with IC(50,GABA) values of 300+/-50microM and 5.0+/-0.7mM, respectively. Binding of CTZ to [3H]-CTZ labeled sites was characterized by IC(50,CTZ) values of 0.16+/-0.03muM ([BMI]=300microM) and 7.0+/-0.5microM ([BMI]=3mM). Binding of the diastereomeric fraction [3H]-(3R,1'S,4'S,5'R+3S,1'R,4'R,5'S)-CTZ induced by 3mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC(50,CTZ)=80+/-15nM and IC(50,GABA)=13+/-3mcapital EM, Cyrillic. In the absence of BMI, CTZ (1mM) significantly decreased GABA-induced enhancement of [3H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding.

Neuroprotective effect of L-kynurenine sulfate administered before focal cerebral ischemia in mice and global cerebral ischemia in gerbils.

Excessive stimulation of N-methyl-D-aspartate (NMDA) receptors during ischemia contributes to apoptotic and excitotoxic nerve cell death. Kynurenic acid is a selective antagonist at the glycine co-agonist site of the NMDA receptor complex at low concentration, and it is a broad-spectrum excitatory amino acid receptor blocker at high concentration. Kynurenic acid provides neuroprotection in animal models of cerebral ischemia only at very high doses as it hardly crosses the blood-brain barrier. The neuroprotective effect of L-kynurenine sulfate, a precursor of kynurenic acid, was therefore studied because L-kynurenine readily crosses the blood-brain barrier. L-kynurenine sulfate was administered 15 min before permanent focal cerebral ischemia produced by electrocoagulation of the distal middle cerebral artery in mice. L-kynurenine sulfate induced a small decrease in the surface area of the brain infarction (10%, P<0.05) at 30 mg/kg i.p., and it caused strong reductions in infarct size (24-25%, P<0.01) at 100 and 300 mg/kg i.p. Treatment of gerbils with L-kynurenine sulfate at 300 mg/kg i.p. 2 h before a 3-min bilateral carotid occlusion decreased (40%, P<0.01) the pyramidal cell loss in the CA1 area of the hippocampus. Furthermore, L-kynurenine sulfate inhibited the ischemia-induced hypermotility (77%, P<0.001), and decreased (50%, P<0.01) the ischemia-induced deterioration of spontaneous alternation, a measure of spatial memory, without altering the rectal temperature. In conclusion, the administration of L-kynurenine can elevate the brain concentration of kynurenic acid to neuroprotective levels, suggesting the potential clinical usefulness of L-kynurenine for the prevention of neuronal loss.