Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7638936 | Journal of Trace Elements in Medicine and Biology | 2018 | 9 Pages |
Abstract
Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe.
Keywords
N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediaminewater soluble tetrazoliumZnT4′,6-diamidin-2-phenylindoleWSTDMEMpNPPZIPFCSSRBFAASDAPITPENCaco-23-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideFLIMMTTp-nitrophenyl phosphateALPAlkaline phosphataseFörster resonance energy transferFRETTemanalysis of varianceANOVAZinc transporterfetal calf serumsulforhodamine BFlame atomic absorption spectrometryInductively-coupled plasma mass spectrometryICP-MSlethal concentrationZincDulbecco’s modified eagle’s mediumSEMScanning electron microscopyTransmission electron microscopyfluorescence lifetime imaging microscopy
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Maria Maares, Claudia Keil, Susanne Thomsen, Dorothee Günzel, Burkhard Wiesner, Hajo Haase,