Growth plate matrix vesicle biogenesis. The role of intracellular calcium.

Isolated bovine growth plate chondrocytes suspended in a synthetic cartilage buffer containing 10(-7) to 10(-3) M concentrations of calcium were treated with 1 microM ionomycin to induce changes in intracellular ionized calcium as measured by Fura-2 fluorescence. An increase in intracellular calcium of 10(-6) to 10(-4) M calcium resulted in the secretion of vesicles that contain alkaline phosphatase. An increase in intracellular calcium above 10(-4) M resulted in nonspecific cell fragmentation because of cell damage. Electron microscopy of the secreted vesicles demonstrated that their structure resembled matrix vesicles. Isolated cells treated with ionomycin (synthetic cartilage buffer with 10(-6) M calcium) demonstrated intact cell membranes, normal intracellular architecture, and numerous plasma membrane processes undergoing vesiculation. The plasma membrane of these isolated cells and the isolated vesicles demonstrated a positive stain for alkaline phosphatase. Fresh growth plate tissue demonstrated plasma membrane staining for alkaline phosphatase at the lower proliferative and upper hypertrophic cell zone, and an increasing number of alkaline phosphatase stained matrix vesicles in the matrix of the lower hypertrophic cell zone. The data indicate that an increase in intracellular calcium in the lower proliferative and hypertrophic cell zones of the growth plate induces the secretion of plasma membrane derived matrix vesicles.