Measurement of intracellular free zinc concentrations accompanying zinc-induced neuronal death

Toxic zinc influx may contribute to selective neuronal death after transien t global ischemia. We previously used the high-affinity (K-D = 27 nM) fluor escent dye mag-fura-5 to detect initial increases in neuronal intracellular free Zn2+ ([Zn2+](i)) associated with brief Zn2+ exposure. Here we used th e specific low-affinity Zn2+ indicator Newport Green (K-D = 1 mu M) to meas ure the peak levels of [Zn2+](i) attained during prolonged, toxic exposures to extracellular Zn2+. Murine cortical cell cultures exposed for 5-10 min to 300 mu M Zn2+ in the presence of kainate or elevated extracellular K+ de veloped widespread neuronal death over the next 24 hr. Such Zn2+ exposure u nder depolarizing conditions was accompanied by a large increase in [Zn2+]( i) reaching several hundred nanomolar, which gradually recovered over the n ext 20-40 min after termination of Zn2+ exposure. Both the level of [Zn2+]( i) elevation and the extent of subsequent neuronal death depended on the co ncentration of extracellular Zn2+ between 30 mu M and 1 mM. In contrast, ex posure to 300 mu M Zn2+ in the presence of 300 mu M NMDA resulted in little increase in [Zn2+](i) and little neuronal death, suggesting that NMDA rece ptor-gated channels are less important as a route of toxic Zn2+ entry than voltage-gated calcium channels.