Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.
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.
Chao
A C
AC
Department of Medicine, University of California, San Francisco 94143.
Dix
J A
JA
Sellers
M C
MC
Verkman
A S
AS
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
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins