Purinergic receptor antagonism prevents cold preservation-induced cell death independent of cellular ATP levels.
BACKGROUND
Purinergic (P2Y) receptors play an important role in intracellular Ca(2+) regulation in hepatocytes. Prevention of mitochondrial Ca(2+) (mCa(2+)) overload during ischemic conditions prevents cellular cell death during the early reperfusion period. P2Y antagonists are cytoprotective in other settings. We studied the effect of P2Y receptor antagonism on mitochondrial associated cell death during the period of cold storage.


METHODS
HepG2 cells were stored in UW with or without 300 muM reactive blue 2 (RB2) or 10 muM ruthenium red (RR) under either normoxic-hypothermic or hypoxic-hypothermic conditions. Cytoplasmic cytochrome c levels were studied by transfection of cytochrome c-GFP. Immunofluorescence determined the intracellular, spatio-temporal distribution of Bax, and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining was used to evaluate cell death. Intracellular compartmental ATP levels were assayed by transfecting with luciferase vectors specific for cytoplasm (PcDNA3-luciferase-LL/V) and mitochondria (PcDNA3-COX8-luciferase).


RESULTS
Bax translocation to the mitochondria occurred immediately following cold storage and was followed by cytochrome c-GFP redistribution to the cytosol during rewarming. RB2 treatment significantly attenuated Bax translocation, cytochrome c-GFP redistribution, and cell death following both storage conditions. Both RR and RB2 provided cytoprotection despite ongoing cytoplasmic ATP consumption during cold ischemia.


CONCLUSIONS
These data indicate that the cytoprotective effects of mCa(2+) uptake inhibition and P2Y receptor antagonism are independent of cytoplasmic ATP levels during cold ischemia.
Anderson
Christopher D
CD
Division of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232-4753, USA.
Pierce
Janene
J
Nicoud
Ian B
IB
Belous
Andrey E
AE
Jones
Christopher M
CM
Chari
Ravi S
RS
D000255:Adenosine Triphosphate / Q000032:analysis*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D003080:Cold Temperature; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D006801:Humans; D008928:Mitochondria / Q000737:chemistry; D021381:Protein Transport; D058919:Purinergic P2 Receptor Antagonists; D012430:Ruthenium Red / Q000494:pharmacology; D014021:Tissue Preservation / Q000379:methods*; D014227:Triazines / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D058919:Purinergic P2 Receptor Antagonists; D014227:Triazines; D051028:bcl-2-Associated X Protein; D012430:Ruthenium Red; C012842:Cibacron Blue F 3GA; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D048049:Extracellular Signal-Regulated MAP Kinases