Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70.
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-kappaB (NF-kappaB). Various pharmacologic inhibitors of the NF-kappaB 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.
Roesslein
Martin
M
Anaesthesiologische Universitaetsklinik, Hugstetterstrasse 55, D-79106 Freiburg, Germany.
Schibilsky
David
D
Muller
Laurent
L
Goebel
Ulrich
U
Schwer
Christian
C
Humar
Matjaz
M
Schmidt
Rene
R
Geiger
Klaus K
KK
Pahl
Heike L
HL
Pannen
Benedikt H J
BH
Loop
Torsten
T
D017209:Apoptosis / Q000187:drug effects*; D017252:CD3 Complex; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000235:genetics*; D018869:Heat-Shock Response / Q000187:drug effects; D006801:Humans; D020011:Protective Agents / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects*; D013874:Thiopental / Q000494:pharmacology*
D017252:CD3 Complex; D018840:HSP70 Heat-Shock Proteins; D020011:Protective Agents; D012333:RNA, Messenger; D013874:Thiopental