Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn2+ indicators, we showed that intracellular regions of Zn2+ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn2+ chelator TPEN, indicating that the local Zn2+ fluorescence represented free Zn2+ localized to the ER in the basal condition. Blockade of the ER Ca2+ pump by thapsigargin produced a steady increase of intracellular Zn2+. Furthermore, we determined that the thapsigargin-induced Zn2+ increase was not dependent on extracellular Ca2+ or extracellular Zn2+, suggesting that it was of intracellular origin. The applications of caged IP3 or IP3-3Kinase inhibitor (to increase available IP3) produced a significant increase in intracellular Zn2+.Taken together, these results suggest that Zn2+ is sequestered into thapsigargin/IP3-sensitive stores and is released upon agonist stimulation.Zn2+ is an important structural and functional component in many cellular proteins and enzymes. As such, Zn2+ levels are normally tightly regulated, limiting the extent of cytosolic labile (or free) Zn2+ concentrations [1,2]. For example, levels of free Zn2+ are several orders of magnitude less than that of Ca2+ [3]. Zn2+ may act as a cellular messenger in physiological and cytotoxic signaling, and the changes in Zn2+ homeostasis have a fundamental effect in cell function [4,5]. Many studies have shown the accumulation of excessive Zn2+ to precede cell death or neurodegeneration in response to cytotoxic stress [6,7]. To characterize Zn2+-mediated signaling pathways or Zn2+-induced cytotoxicity, it is important to determine the source(s) of intracellular free Zn2+ in response to specific stimuli or injury.The endoplasmic reticulum (ER) is an intracellular organelle that has been shown to sequester Ca2+ from the cytosol by means of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase