Martin Roesslein1, Christian Froehlich2, Frank Jans3, Tobias Piegeler4, Ulrich Goebel2, Torsten Loop2. 1. Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany. Electronic address: martin.roesslein@uniklinik-freiburg.de. 2. Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany. 3. Dept. of Anaesthesiology and Critical Care Medicine, Ziekenhuis Oost-Limburg, Genk and Biomedical Research Institute, UHasselt, Diepenbeek, Belgium. 4. Institute of Anaesthesiology, University Hospital Zurich, Switzerland; Dept. of Anesthesiology, University of Illinois at Chicago, USA.
Abstract
AIMS: 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 hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response. MAIN METHODS: Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed 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. KEY FINDINGS: Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20-32] % vs. 12 [9-15] % for dobutamine 0.1 mM and 7 [5-12] % for dobutamine 0.5 mM, 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 0.1 mM and 0.20 [0.19-0.23] for Dobutamine 0.5 mM, 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.
AIMS: 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 hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response. MAIN METHODS: Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed 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. KEY FINDINGS: Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20-32] % vs. 12 [9-15] % for dobutamine 0.1 mM and 7 [5-12] % for dobutamine 0.5 mM, 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 0.1 mM and 0.20 [0.19-0.23] for Dobutamine 0.5 mM, 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.