2-2--(hydroxynitrosohydrazono)bis-ethanamine and zinquin

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function.

Topics: Animals; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Flow Cytometry; Fluorescence; Fluorescent Dyes; Homeostasis; Islets of Langerhans; Nitric Oxide; Nitric Oxide Donors; Quinolones; Rats; Rats, Wistar; Tosyl Compounds; Triazenes; Zinc

We previously described that NO. leads to destruction of ZnS clusters and release of Zn2+ from various proteins including zinc finger transcription factors. To assess the relevance in living cells, we investigated, whether exogenous NO. leads to an increase of cytoplasmic and nuclear free Zn2+. L929 cells, mouse splenocytes, or rat aorta endothelial cells were labeled with Zinquin-E, a Zn2+-specific fluorophore, and were treated with two different spontaneous NO donors, S-nitrosocysteine or DETA/NO. Both NO donors strongly increased the Zn2+-dependent fluorescence in the cellular cytosol and also in nuclei as compared to controls. NO-dependent Zn2+ release in splenocytes was quantitated by flow cytometry. These results show for the first time, that nitrosative stress mediates intracellular and intranuclear Zn2+ release which may be relevant in altering gene expression patterns.

Topics: Animals; Aorta; Cell Line; Cell Nucleus; Cells, Cultured; Cysteine; Cytoplasm; Endothelium, Vascular; Fluorescent Dyes; Mice; Nitric Oxide; Quinolones; Rats; S-Nitrosothiols; Spleen; Tosyl Compounds; Triazenes; Zinc