RECORDING OF MITOCHONDRIAL TRANSMEMBRANE POTENTIAL AND VOLUME IN CULTURED RAT OSTEOCLASTS BY CONFOCAL LASER-SCANNING MICROSCOPY

Osteoclasts are multinuclear bone-resorbing cells which contain abunda nt mitochondria. Morphological studies have suggested that a correlati on may exist between mitochondrial concentration and bone resorption b y osteoclasts. However, investigation of mitochondrial transmembrane p otential (Delta Psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these char acteristics by flow cytometric analysis. In this study, we have used c onfocal laser scanning microscopy after loading the cells with Rhodami ne 123 and 10-nonyl Acridine Orange to record mitochondrial Delta Psi and volume, respectively, in isolated rat osteoclasts cultured on bovi ne bone slices. Optimal staining conditions were found to be 10 mu g m l(-1) for 40 min for Rhodamine, and 1 mu M for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape s eemed to reflect bone resorption and migratory functions, were identif ied depending on their shape and on the distribution of the two dye pr obes. 'Round-shaped' osteoclasts had significantly higher mitochondria l Delta Psi and volume in the apical regions than in the basolateral p ortions (p < 0.00001). In contrast, mitochondrial Delta Psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in b oth these regions (p > 0.05). Our results indicate that there is an ap ical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.