Chloral hydrate disrupts mitosis by increasing intracellular free calcium

In examining how chloral hydrate affects mitosis, we found that extracellular application of 0.1% chloral hydrate produced an abrupt rise in cytosolic free Ca2+. Digitized fluorescence microscopy of Fura-2-loaded, mitotic and interphase PtK cells revealed that Ca2+ rose 15 s after chloral hydrate application, peaked within 1 min at a concentration two- to sevenfold above the basal level and then slowly dropped. Bathing cells in 0.1% chloral hydrate caused metaphase spindles to shorten, starting in 1-2 min, and inhibited spindle elongation without affecting chromosome-to-pole movement during anaphase, as determined by phase-contrast observation of living cells. Spindle elongation and chromosome movement were unaffected by intracellular injection of 7.5% chloral hydrate. Extensive mitotic microtubule breakdown occurred after cells were bathed for 7 min in 0.1% chloral hydrate, while interphase microtubules were unaffected as determined by immunofluorescence. The chloral hydrate-induced microtubule breakdown and metaphase spindle shortening were prevented by 10 mM-CoCl2, which has previously been shown to block Ca2+ influx and to stabilize microtubules in vitro. These results imply that disruption of mitotic spindle function and structure by chloral hydrate is due to a rise in cytosolic free Ca2+, and also indicate that mitotic microtubules are more Ca2+-labile than interphase microtubules.