Thiopental inhibits tumor necrosis factor α-induced activation of nuclear factor κB through suppression of IκB kinase activity

Abstract

Background: Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury and is associated with immunosuppressive effects. The authors have recently reported that thiopental inhibits activation of nuclear factor (NF) κB, a transcription factor implicated in the expression of many inflammatory genes. Thus, it was the aim of the current study to examine the molecular mechanism of this inhibitory effect. Methods: The authors tested γ-aminobutyric acid (GABA), the GABAA antagonist bicuculline, and the GABAB antagonist dichlorophenyl-methyl-amino-propyl-diethoxymethyl-phosphinic acid (CGP 52432) in combination with thiopental for their influence on the activation of NF-κB. In addition, they investigated the direct effect of thiopental on activated NF-κB DNA binding activity. These experiments were conducted in Jurkat T lymphocytes using electrophoretic mobility shift assays. The presence of the phosphorylated and dephosphorylated NF-κB inhibitor IκBα (Western blotting) and IκB kinase activity were studied in Jurkat T cells and human CD3+ T lymphocytes. In addition, the authors tested the effect of the structural barbiturate analog pairs thiopental-pentobarbital and thiamylal-secobarbital and of thiopental in combination with the thio-group containing chemical dithiothreitol on the activation of NF-κB. Results: GABA did not inhibit NF-κB activation, and the GABAA and GABAB antagonists bicuculline and CGP did not diminish the thiopental-mediated inhibitory effect on NF-κB activation. Thiopental did not inhibit activated NF-κB directly in a cell-free system. The phosphorylation of IκBα was prevented after incubation with 1,000 μg/ml thiopental. The same concentration of thiopental also inhibited IκB kinase activity in tumor necrosis factor-stimulated Jurkat T cells and human CD3+ T lymphocytes (60% suppression, P < 0.05 vs. tumor necrosis factor α alone). Thiobarbiturates (4 ± 10-3 M) inhibited NF-κB activity, whereas equimolar concentrations of the structural oxyanalogs did not. Preincubation of thiopental with dithiothreitol diminished the inhibitory effect. Conclusion. Thiopental-mediated inhibition of NF-κB activation is due to the suppression of IκB kinase activity and depends at least in part on the thio-group of the barbiturate molecule.