Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.
Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.
Fulceri
R
R
Istituto di Patologia Generale, University of Siena, Italy.
Bellomo
G
G
Mirabelli
F
F
Gamberucci
A
A
Benedetti
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology*; D001127:Arginine Vasopressin / Q000494:pharmacology; D001150:Arsenazo III; D001693:Biological Transport, Active; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology*; D016257:Fura-2; D006873:Hydroquinones / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D003012:Microbial Collagenase; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D006873:Hydroquinones; D001127:Arginine Vasopressin; D001150:Arsenazo III; C019359:2,5-di-tert-butylhydroquinone; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000001:Calcimycin; D000968:Antimycin A; D000255:Adenosine Triphosphate; D003012:Microbial Collagenase; D002118:Calcium; D016257:Fura-2