Abstract
Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-κB (NF-κB). Various pharmacologic inhibitors of the NF-κB pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3+ T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed by Western blot and flow cytometry after fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response, and it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.
Footnotes
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M.R. and D.S. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.133108.
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ABBREVIATIONS: NF-κB, nuclear factor-κB; HSR, heat shock response; HSF, heat shock factor; HSE, heat shock element; hsp, heat shock protein; hsp, heat 70-kDa shock protein; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; FL, fluorescence channel; PE, phycoerythrin; 7-AAD, 7-amino-actinomycin D; IκB, inhibitor of nuclear factor-κB; ANOVA, analysis of variance; Hsc, heat shock cognate protein.
- Received October 16, 2007.
- Accepted January 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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