Article

Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70.

Anaesthesiologische Universitaetsklinik, Hugstetterstrasse 55, D-79106 Freiburg, Germany.
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.89). 05/2008; 325(1):217-25. DOI: 10.1124/jpet.107.133108
Source: PubMed

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-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.

0 Bookmarks
 · 
82 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Epilepsy refers to a cluster of neurological diseases characterized by seizures. Although many forms of epilepsy have a well-defined immune etiology, in other forms of epilepsy an altered immune response is only suspected. In general, the hypothesis that inflammation contributes to seizures is supported by experimental results. Additionally, antiepileptic maneuvers may act as immunomodulators and anti-inflammatory therapies can treat seizures. Triggers of seizure include a bidirectional communication between the nervous system and organs of immunity. Thus, a crucial cellular interface protecting from immunological seizures is the blood-brain barrier (BBB). Here, we summarize recent advances in the understanding and treatment of epileptic seizures that derive from a non-neurocentric viewpoint and suggest key avenues for future research.
    Trends in Neurosciences 12/2013; · 13.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesised that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response. Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analysed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile. Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20-32] % vs. 12 [9-15] % for dobutamine 0.1mM and 7 [5-12] % for dobutamine 0.5mM, p<0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40-0.48] vs. 0.32 [0.27-0.39] for Dobutamine 0.1mM and 0.20 [0.19-0.23] for Dobutamine 0.5mM], p<0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32-36] % vs. 18 [16-24] %) and caspase-3-like activity (0.21 [0.19-0.23] vs. 0.16 [0.13-0.17], p<0.05) SIGNIFICANCE: Dobutamine protects from apoptotic cell death via the induction of Hsp70.
    Life sciences 01/2014; · 2.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP-depletion, loss of ion homeostasis, membrane depolarisation, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor eEF2 that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP-content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cyclic adenosine monophosphate increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP-availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP-content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.
    Journal of Pharmacology and Experimental Therapeutics 09/2013; · 3.89 Impact Factor

Full-text

View
32 Downloads
Available from
May 26, 2014