title: Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.
abstract: The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells.
authors: Department of Medicine, University of California, San Francisco 94143.
authors: A C
authors: AC
authors: Chao
authors: J A
authors: JA
authors: Dix
authors: M C
authors: MC
authors: Sellers
authors: A S
authors: AS
authors: Verkman
mesh_terms: D000818:Animals; D001693:Biological Transport, Active; D002460:Cell Line; D002478:Cells, Cultured; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism* / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D010477:Perfusion / Q000295:instrumentation / Q000379:methods
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins