Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.
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.
Thompson
R B
RB
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, MD 21201, USA.
Whetsell
W O
WO
Maliwal
B P
BP
Fierke
C A
CA
Frederickson
C J
CJ
D000818:Animals; D000831:Animals, Newborn; D016880:Anisotropy; D015374:Biosensing Techniques / Q000379:methods*; D002256:Carbonic Anhydrases / Q000494:pharmacology*; D004558:Electric Stimulation; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D008856:Microscopy, Fluorescence / Q000379:methods*; D009924:Organ Culture Techniques; D010069:Oxadiazoles / Q000138:chemical synthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D013449:Sulfonamides / Q000138:chemical synthesis; D015032:Zinc / Q000378:metabolism*
C407582:4-(aminosulfonyl)-7-(ethanolamino)-2,1,3-benzoxadiazole; D010069:Oxadiazoles; D011994:Recombinant Proteins; D013449:Sulfonamides; D002256:Carbonic Anhydrases; D015032:Zinc