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

BACKGROUND: Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP(3)-gated thapsigargin sensitive intracellular calcium (Ca(2+)) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn(2+)) that has been reported to play a role in cell signaling. RESULTS: In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn(2+ )indicators, we showed that intracellular regions of Zn(2+ )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 Zn(2+ )chelator TPEN, indicating that the local Zn(2+ )fluorescence represented free Zn(2+ )localized to the ER in the basal condition. Blockade of the ER Ca(2+ )pump by thapsigargin produced a steady increase of intracellular Zn(2+). Furthermore, we determined that the thapsigargin-induced Zn(2+ )increase was not dependent on extracellular Ca(2+ )or extracellular Zn(2+), suggesting that it was of intracellular origin. The applications of caged IP(3 )or IP(3)-3Kinase inhibitor (to increase available IP(3)) produced a significant increase in intracellular Zn(2+). CONCLUSIONS: Taken together, these results suggest that Zn(2+ )is sequestered into thapsigargin/IP(3)-sensitive stores and is released upon agonist stimulation.