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

Abstract

Background: Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP3-gated thapsigargin sensitive intracellular calcium (Ca2+) 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 (Zn2+) that has been reported to play a role in cell signaling.

Results: 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+.

Conclusions: Taken together, these results suggest that Zn2+ is sequestered into thapsigargin/IP3-sensitive stores and is released upon agonist stimulation.

Figure 1

Co-localization of intracellular Zn²⁺ and ER fluorescence. Images of cultured cortical neurons co-labeled with the intracellular fluorescent Zn²⁺ indicators and a live cell marker for the ER. A. Fluorescent Zn²⁺ indicator Newport Green AM co-localizes with the ER marker ER-Tracker Red. B. Fluorescent Zn²⁺ indicator ZinPyr-1 co-localizes with the ER marker ER-Tracker Red. Cells were incubated with 10 μM of the specified fluorescent Zn²⁺ indicator and 1 μM ER-Tracker Red then imaged using a LSM510 confocal microscope equipped with a 100x/1.3NA objective.

Figure 2

Effect of Zn²⁺ chelation. The graph indicates the fluorescent response of Newport Green (green line) and ER-Tracker Red (red line) in unstimulated neurons upon TPEN (10 μM) exposure, data points represent the mean ± SD of n = 5 trials. The quenching of Newport Green fluorescence with TPEN indicates the presence of free Zn²⁺ in the basal condition.

Figure 3

Thapsigargin-induced Zn²⁺ release in cultured cortical neurons. A. The graph shows the increases in fluorescence intensity of cells in response to the treatment with thapsigargin (2 μM). Data points represent the mean ± SD of n = 3 trials. Cells were labeled with Newport Green (10 μM). B. Representative fluorescent images of cortical neurons loaded with Newport Green before and after exposure to thapsigargin.

Figure 4

Summary of Zn²⁺ transients induced by thapsigargin in cortical neurons. The bar graphs show peak fluorescence after the treatment with thapsigargin alone (n = 6), thapsigargin plus TPEN (10 μM; n = 6), thapsigargin plus CaEDTA (1 mM; n = 3), and thapsigargin in the medium without added Ca²⁺ (n = 4). Data points represent the mean ±SD.

Figure 5

IP₃ stimulation results in the release of intracellular Zn²⁺. A. The photo-uncaging of caged IP₃ (2 μM) in brain hippocampal neurons loaded with the Zn²⁺ fluorescent indicator Newport Green results in an increase of cytosolic Zn²⁺. Scale bar = 20 μm and the images a, b, and corresponding to points in B. B. The graph shows a change of fluorescence in response to IP₃ stimulation. C. A change of fluorescence in responses to the treatments of the IP₃-3K inhibitor N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine (20 μM) and TPEN (10 μM) in cells loaded with Newport Green. D. Bar graph shows the average response of Newport Green to the IP₃K-inhibitor (n = 4). The application of TPEN at the peak of fluorescence resulted in significant (n = 2) fluorescence quenching, demonstrating that the fluorescence elevation was due to increased free Zn²⁺.