Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts.
Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2(+)-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 +/- 0.2 microM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mt increase, although it did not affect the [Ca2+]c transient. Basal [Ca2+]c and [Ca2+]mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 +/- 1% in normal cells to 19 +/- 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.
Padua
R A
RA
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
Baron
K T
KT
Thyagarajan
B
B
Campbell
C
C
Thayer
S A
SA
D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014475:Uncoupling Agents / Q000494:pharmacology
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D003574:Cytochrome c Group; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014475:Uncoupling Agents; C060014:apoaequorin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000331:Aequorin; D001920:Bradykinin; D002118:Calcium