title: Methods for studying synaptosomal copper release.
abstract: Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilson's, Alzheimer's, and probably in prion protein diseases like Creutzfeld-Jakob's disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca2+-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu2+ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis-(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu2+ (apparent dissociation constant Kd=0.43 +/- 0.07 microM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca2+ and Mg2+, unlike most of the other dyes examined. In addition, Zn2+ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a Kd of 50 +/- 2.5 microM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry).
authors.affiliation: Institut für Neuropathologie, Ludwig-Maximilians-Universität München, Marchioninistr. 17, 81377 München, Germany. alexander.hopt@charite.de
authors.forename: Alexander
authors.initials: A
authors.lastname: Hopt
authors.forename: Stefan
authors.initials: S
authors.lastname: Korte
authors.forename: Herbert
authors.initials: H
authors.lastname: Fink
authors.forename: Ulrich
authors.initials: U
authors.lastname: Panne
authors.forename: Reinhard
authors.initials: R
authors.lastname: Niessner
authors.forename: Reinhard
authors.initials: R
authors.lastname: Jahn
authors.forename: Hans
authors.initials: H
authors.lastname: Kretzschmar
authors.forename: Jochen
authors.initials: J
authors.lastname: Herms
mesh_terms: D000818:Animals; D002417:Cattle; D002463:Cell Membrane Permeability / Q000502:physiology; D019439:Chromaffin Cells / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000032:analysis* / Q000502:physiology*; D013058:Mass Spectrometry / Q000379:methods; D011166:Porphyrins / Q000032:analysis; D013574:Synaptosomes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011166:Porphyrins; C016136:tetraphenylporphine sulfonate; D003300:Copper