Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes

Abstract Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn 2+ . Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn 2+ with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn 2+ as the chondrocytes mature. Intracellular labile Zn 2+ was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn‐chelator toluenesulfonamidoquinoline (TSQ) and imaged by multi‐photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin. Short‐term exposure to Zn 2+ , both in the presence and absence of pyrithione resulted in significant increases in cytosolic Zn 2+ . Treatment with the membrane‐permeant Zn 2+ chelator TPEN rapidly reduced the levels of labile Zn 2+ and triggered apoptosis. Cytosolic Zn 2+ levels were significantly reduced following 24‐h incubations with known inducers of chondrocyte apoptosis. The loss of intracellular Zn 2+ was accompanied by a significant reduction in the cytosolic metal‐binding protein metallothionein. Examination of Zn 2+ ‐treated cells with MPLSM showed uniformly higher zinquin fluorescence. Treatment of Zn 2+ ‐loaded cells with TPEN quenched zinquin fluorescence confirming that the observed fluorescence in chondrocytes is due to the presence of intracellular Zn 2+ . A dose‐dependent increase in zinquin fluorescence was observed in cells treated with a range of Zn 2+ concentrations. Short‐term treatment of cultured chondrocytes with apoptosis‐inducing chemicals resulted in transient increases in intracellular labile Zn 2+ . These results indicate that Zn 2+ is mobilized from intracellular binding sites in the early stages of chondrocyte apoptosis and is subsequently lost from the cells. The early mobilization of Zn 2+ provides a mechanism for its movement to matrix vesicles and the extracellular matrix. J. Cell. Biochem. 88: 954–969, 2003. © 2003 Wiley‐Liss, Inc.