Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate

In the companion article, we reported that the local phosphate (Pi) concent ration triggers apoptosis in epiphyseal chondrocytes. The goal of the curre nt investigation was to evaluate the apoptotic process in relationship to t he energy status of cells in the growth plate. For these studies, we used s ections of the adolescent growth plate, as well as cells isolated from the tissue. We found that there was a maturation-dependent loss of mitochondria l function in growth plate chondrocytes and these cells generated energy by glycolysis. Since treatment with the uncoupler 2,4-dinitrophenol as well a s the site-specific inhibitors antimycin A and rotenone failed to elicit a further increase in the activity of the glycolytic pathway, we concluded th at oxidative metabolism was minimum in these cells. Flow cytometric studies of growth plate cells and confocal microscopy of growth plate sections usi ng the mitochondrial probes Rh123 and DiOC(6)(3) provided unequivocal evide nce that there was loss of mitochondrial membrane potential in hypertrophic cells. Furthermore, the intrinsic fluorescence of the flavoprotein lipoami de dehydrogenase complex of the electron transport chain revealed that the mitochondria were in an oxidized state. Finally, we assessed Bcl-2 expressi on in these cells. Although immunohistochemical and Western blot analysis s howed that the chick cells contained a low level of the anti-apoptotic prot ein Bcl-2, reverse transcription-polymerase chain reaction (RT-PCR) analysi s indicated that transcripts were present in chondrocytes. Based on these o bservations, we suggest that terminally differentiated chondrocytes undergo a maturation-dependent loss of mitochondrial function. In concert with the low expression of Bcl-2, they become sensitive to signals for programmed c ell death. We hypothesize that Pi triggers apoptosis in these energy-compro mised cells by promoting a mitochondrial membrane transition, thereby induc ing the death process. (C) 1999 Wiley-Liss, Inc.