Heterogeneity of chondrocyte mitochondria: A study of the Ca concentration and density banding characteristics of normal and rachitic cartilage

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca²⁺ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5–2.0 M) in the presence of inhibitors of Ca²⁺ transport (ruthenium red and 5,5′-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca²⁺ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca²⁺ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca²⁺ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca²⁺ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca²⁺ concentration necessary for initiation of the calcification mechanism was 4 μmol/mg protein.