Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro.
OBJECTIVE
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.


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.


RESULTS
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).


CONCLUSIONS
Dobutamine protects from apoptotic cell death via the induction of Hsp70.
Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany. Electronic address: martin.roesslein@uniklinik-freiburg.de.
Martin
M
Roesslein
Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany.
Christian
C
Froehlich
Dept. of Anaesthesiology and Critical Care Medicine, Ziekenhuis Oost-Limburg, Genk and Biomedical Research Institute, UHasselt, Diepenbeek, Belgium.
Frank
F
Jans
Institute of Anaesthesiology, University Hospital Zurich, Switzerland; Dept. of Anesthesiology, University of Illinois at Chicago, USA.
Tobias
T
Piegeler
Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany.
Ulrich
U
Goebel
Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany.
Torsten
T
Loop
D058665:Adrenergic beta-1 Receptor Agonists / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015152:Blotting, Northern; D045744:Cell Line, Tumor; D019610:Cytoprotection / Q000187:drug effects*; D004280:Dobutamine / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D000097782:NF-kappaB-Inducing Kinase
D058665:Adrenergic beta-1 Receptor Agonists; D004280:Dobutamine; D018840:HSP70 Heat-Shock Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D000097782:NF-kappaB-Inducing Kinase
Apoptosis; Dobutamine; Dobutamine hydrochloride (PubChem CID: 65324); Heat shock protein 70; Heat shock response; Protection