Effect of cold preservation on intracellular calcium concentration and calpain activity in rat sinusoidal endothelial cells.
This study was performed to determine the role of intracellular calcium concentration and calpain activity on the cellular events that occur in rat sinusoidal endothelial cells (SEC) in the cold. Intracellular calcium concentrations were measured in isolated cold preserved rat SEC. Dantrolene or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was added in some studies. In other studies, calpain activity and m-calpain and mu-calpain expression were measured during cold preservation in the presence or absence of calpain inhibitors. The effect of addition of dantrolene to preservation solutions on function of whole livers after preservation was determined. Cold preservation caused an increase in intracellular calcium concentration first detected at 1 hour of preservation. This was associated with cell rounding and actin disassembly. Dantrolene and BAPTA-AM prevented the increase in intracellular calcium concentration and reduced cell rounding and actin disassembly. Cold preservation also resulted in increased calpain activity and expression on SEC. Calpain expression was reduced by dantrolene. Calpain inhibitors N-acetyl-leu-leu-norleucinal (ALLN) and N-acetyl-leu-leu-methioninal (ALLM) reduced calpain activity and expression and restored SEC cell shape and actin morphology. Dantrolene improved function of livers preserved in Eurocollins (EC) solution when tested on the isolated perfused rat liver (IPRL). In conclusion, exposure of SEC to cold results sequentially in elevated intracellular calcium concentration, increased calpain activity, and actin disassembly.
Upadhya
G Aravinda
GA
Department of Surgery, Washington University in St. Louis, MO 63110, USA.
Topp
Stefan A
SA
Hotchkiss
Richard S
RS
Anagli
John
J
Strasberg
Steven M
SM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002403:Cathepsins / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D015925:Cryopreservation; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003620:Dantrolene / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008102:Liver Circulation; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism; D009994:Osmolar Concentration; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017207:Rats, Sprague-Dawley
D002121:Calcium Channel Blockers; D002614:Chelating Agents; D009097:Multienzyme Complexes; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D002403:Cathepsins; D002154:Calpain; D003546:Cysteine Endopeptidases; C449000:m-calpain; C449002:mu-calpain; D046988:Proteasome Endopeptidase Complex; D003620:Dantrolene; D002118:Calcium