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Inhibition of chondrogenesis and endochondral mineralization in vitro by different calcium channel blockers.

Limb bud mesenchymal cells from mouse embryos grown at high density at the medium/air interphase undergo chondrogenesis and form numerous nodules of mature cartilage. Addition of beta-glycerophosphate (5 mM) induced endochondral mineralization within these nodules. Ca2+ accumulation, matrix formation and alkaline phosphatase activity were recorded for each culture. Treatment with the L-type channel-specific blockers nifedipine and verapamil during the entire culture period caused an inhibition of mineralization. Sequential treatment reduced mineralization only when added during the early part of the culture period in the course of chondrogenesis. In all cases, matrix formation, estimated by alcian blue binding, was concomitantly diminished. Lanthanum acetate, which blocks Ca2+ channels non-specifically, also reduced Ca2+ accumulation in the cultures when added continuously. After sequential treatment, Ca2+ content was only diminished when the cultures were treated in the last part of the culture period during mineralization. Matrix formation was not altered by lanthanum acetate, but alkaline phosphatase was decreased. These results show that chondrogenic differentiation is under control of L-type Ca2+ channels, whereas matrix calcification depends on intracellular Ca2+ accumulation mediated by lanthanum-sensitive Ca2+ channels. This points to the importance of intracellular Ca2+ accumulation in the process of endochondral mineralization.