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.
Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo, São Paulo, SP, 05508-900, Brazil. alicia@iq.usp.br
Alicia J
AJ
Kowaltowski
Ricardo G
RG
Cosso
Claudia B
CB
Campos
Gary
G
Fiskum
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