Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.

摘要

We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and a fluorescent aryl sulfonamide inhibitor of the enzyme, ABD-N. The apoenzyme and ABD-N in the absence of Zn(II) exhibit weak, reddish fluorescence typical of the ABD-N alone; when Zn(II) is added it binds to the apoenzyme (K(D) = 4 pM), which strongly promotes binding of ABD-N to the holoenzyme (K(D) = 0.9 microM). Binding of ABD-N to the holoenzyme results in a 9-fold increase in apparent quantum yield, significant blue shifts in excitation and emission, an increase in average fluorescence lifetime, a 4-fold increase in the ratio of intensities at 560 and 680 nm, and a large increase in anisotropy. Prior to stimulation, cultures immersed in phosphate-buffered saline with glucose and apoCAII with ABD-N emitted negligible fluorescence, but within 20 s after electrical stimulation a diffuse cloud of greenish fluorescence emerged and subsequently covered most of the culture, indicating release of zinc into the extracellular medium.