Thiopental inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappaB through suppression of kappaB kinase activity.
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) kappaB, 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 gamma-aminobutyric acid (GABA), the GABA(A) antagonist bicuculline, and the GABA(B) antagonist dichlorophenyl-methyl-amino-propyl-diethoxymethyl-phosphinic acid (CGP 52432) in combination with thiopental for their influence on the activation of NF-kappaB. In addition, they investigated the direct effect of thiopental on activated NF-kappaB DNA binding activity. These experiments were conducted in Jurkat T lymphocytes using electrophoretic mobility shift assays. The presence of the phosphorylated and dephosphorylated NF-kappaB inhibitor IkappaBalpha (Western blotting) and IkappaB 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-kappaB.


RESULTS
GABA did not inhibit NF-kappaB activation, and the GABA(A) and GABA(B) antagonists bicuculline and CGP did not diminish the thiopental-mediated inhibitory effect on NF-kappaB activation. Thiopental did not inhibit activated NF-kappaB directly in a cell-free system. The phosphorylation of IkappaBalpha was prevented after incubation with 1,000 microg/ml thiopental. The same concentration of thiopental also inhibited IkappaB kinase activity in tumor necrosis factor-stimulated Jurkat T cells and human CD3+ T lymphocytes (60% suppression, P < 0.05 vs. tumor necrosis factor alpha alone). Thiobarbiturates (4 x 10(-3) m) inhibited NF-kappaB activity, whereas equimolar concentrations of the structural oxyanalogs did not. Preincubation of thiopental with dithiothreitol diminished the inhibitory effect.


CONCLUSIONS
Thiopental-mediated inhibition of NF-kappaB activation is due to the suppression of IkappaB kinase activity and depends at least in part on the thio-group of the barbiturate molecule.
Loop
Torsten
T
Department of Anesthesiology and Critical Care Medicine, University Hospital, Freiburg, Germany.
Humar
Matjaz
M
Pischke
Soeren
S
Hoetzel
Alexander
A
Schmidt
Rene
R
Pahl
Heike L
HL
Geiger
Klaus K
KK
Pannen
Benedikt H J
BH
D018686:Anesthetics, Intravenous / Q000494:pharmacology*; D015153:Blotting, Western; D017252:CD3 Complex / Q000378:metabolism; D002474:Cell-Free System; D004229:Dithiothreitol / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D051550:I-kappa B Kinase; D007202:Indicators and Reagents; D019169:Jurkat Cells; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D018079:Receptors, GABA / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000187:drug effects; D013858:Thiobarbiturates / Q000494:pharmacology; D013874:Thiopental / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology*
D018686:Anesthetics, Intravenous; D017252:CD3 Complex; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D016328:NF-kappa B; D018079:Receptors, GABA; D013858:Thiobarbiturates; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D013874:Thiopental; D004229:Dithiothreitol