title: In vivo fluorescence measurement of Na(+) concentration in the pericryptal space of mouse descending colon.
abstract: A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring colonic crypt function in vivo in mice. Na(+) concentration in the extracellular pericryptal space of descending colon was measured using a low-affinity Na(+)-sensitive fluorescent indicator consisting of an Na(+)-sensitive chromophore (sodium red) and an Na(+)-insensitive chromophore (Bodipy-fl) immobilized on 200-nm-diameter polystyrene beads. The Na(+) indicator beads accumulated in the pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na(+) concentration ([Na(+)]) in the pericryptal space was 491 +/- 62 mM (n = 4). After a 70-min exposure to amiloride (0.25 mM), pericryptal [Na(+)] was reduced to 152 +/- 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [Na(+)] to 204 +/- 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dye into the crypt lumen with a half time of 19.8 +/- 1.0 s, which was increased to 77.9 +/- 6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measure colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The pericryptal space represents the first example of a hypertonic extracellular compartment in mammals that is not created by a countercurrent amplification mechanism.
authors.affiliation: Division of Physiology, Centre for Vascular Biology and Medicine, King's College London, Guy's Campus, London SE1 1UL, United Kingdom.
authors.forename: J R
authors.initials: JR
authors.lastname: Thiagarajah
authors.forename: S
authors.initials: S
authors.lastname: Jayaraman
authors.forename: R J
authors.initials: RJ
authors.lastname: Naftalin
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002138:Calibration; D003106:Colon / Q000378:metabolism* / Q000601:surgery; D003911:Dextrans / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008863:Microspheres; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism*
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; C015219:fluorescein isothiocyanate dextran; D000584:Amiloride; D012964:Sodium; D016650:Fluorescein-5-isothiocyanate