Opposing effects on mitochondrial membrane potential by malonate and levamisole, whose effect on cell-mediated mineralization is antagonistic.

The act of chondrocyte preparation for primary, enchondral, mineralization is associated with a decline in mitochondrial respiration toward the end of the proliferative zone and the hypertrophic zone in the growth plate. Dexamethasone (Dex)-stimulated cultures of rat marrow stroma constitute a differentiation model simulating, in its energy metabolism, chondrocyte mineralization. In this model, early inhibition of succinate dehydrogenase (SDH) enriches the culture with mineralizing cells, whereas levamisole inhibits mineralization. Dex also increases mitochondrial membrane potential in stromal cells, especially on days 7-8 of stimulation. In the present study, suicide inhibition of SDH, by nitropropionic acid (NPA), in Dex-stimulated cells showed a dose-dependent increase in day 21 mineralization; the maximal effect was induced on days 2-4 of stimulation. Mineralization under 2-day-long exposure to NPA showed a similar trend to the previously studied effect of continuous exposure to malonate applied between days 3-11. Unlike malonate, the effect of NPA required its presence in the cultures for only 2 days and resulted in higher mineralization than that seen under 8 days of malonate. NPA delineated a period, days 2/4 to 7/9, in which inhibition of succinate oxidation is necessary to augment mineralization. During this period, NPA also exhibited OPC selection capacity. Early application of levamisole, under conditions previously shown to decrease day 21 mineralization, maintained mitochondrial membrane potential at the beginning of Dex stimulation but decreased or had little effect on it during days 5-10. By contrast, malonate previously found to increase day 21 mineralization decreased the membrane potential at the beginning of Dex stimulation but increased it later on day 7, or during days 5-10. These results indicate that during osteoprogenitor differentiation, before the mineralization stage, a surge in mitochondrial inner membrane potential during late matrix maturation may be a marker that heralds the extracellular matrix mineralization.