Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes.
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.
Sauer
Glenn R
GR
Biology Department, Fairfield University, Fairfield, Connecticut 06430, USA. gsauer@mail.fairfield.edu
Smith
Della M
DM
Cahalane
Matthew
M
Wu
Licia N Y
LN
Wuthier
Roy E
RE
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002413:Cations, Divalent; D002478:Cells, Cultured; D002645:Chickens; D019902:Chondrocytes / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D006132:Growth Plate / Q000737:chemistry / Q000378:metabolism*; D008682:Metatarsal Bones / Q000254:growth & development; D018613:Microscopy, Confocal; D015363:Quinolones; D013977:Tibia / Q000254:growth & development; D014105:Tosyl Compounds; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D015363:Quinolones; D014105:Tosyl Compounds; D015032:Zinc; C084731:zinquin