Effect of Bcl-2 overexpression on mitochondrial structure and function.
Overexpression of the antiapoptotic Bcl-2 protein enhances the uptake of fluorimetric dyes sensitive to mitochondrial membrane potential, suggesting that Bcl-2 changes the mitochondrial proton gradient. In this study, we performed calibrated measurements of mitochondrial respiration, membrane potential, deltapH, and intramitochondrial [K+] in digitonin-permeabilized PC12 and GT1-7 neural cells that either do not express human Bcl-2 (control transfectants) or that were transfected with and overexpressed the human bcl-2 gene to evaluate whether Bcl-2 alters mitochondrial inner membrane ion transport. We found that although Bcl-2-overexpressing cells exhibit higher fluorescence responses to membrane potential, pH, and K+-sensitive dyes, this increased response is due to an enhanced accumulation of these dyes and not an increased mitochondrial membrane potential, deltapH, or [K+]. This result is supported by the presence of equal respiratory rates in Bcl-2+ and Bcl-2- cells. Possible structural alterations in Bcl-2+ mitochondria that could account for increases in fluorescent dye uptake were evaluated using flow cytometry particle sizing and light scattering determinations. These experiments established that Bcl-2-overexpressing mitochondria present both increased volume and structural complexity. We suggest that increased mitochondrial volume and structural complexity in Bcl-2+ cells may be related to many of the effects of this protein involved in the prevention of cell death.
Kowaltowski
Alicia J
AJ
Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo, São Paulo, SP, 05508-900, Brazil. alicia@iq.usp.br
Cosso
Ricardo G
RG
Campos
Claudia B
CB
Fiskum
Gary
G
D000818:Animals; D016923:Cell Death / Q000502:physiology; D002460:Cell Line; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D019254:Genes, bcl-2; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007031:Hypothalamus; D007425:Intracellular Membranes / Q000502:physiology; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure*; D009474:Neurons / Q000166:cytology; D016716:PC12 Cells; D010673:Pheochromocytoma; D011188:Potassium / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D051381:Rats
D019253:Proto-Oncogene Proteins c-bcl-2; D011188:Potassium