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Chloride determination in human lens epithelial cells (B3) by a fluorescent dye
Author(s) -
Chimote Ameet Ajit,
Adragorma C.,
Lauf Peter K.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1337
Subject(s) - nigericin , chemistry , fluorescence , chloride , analytical chemistry (journal) , bromide , iodide , membrane , nuclear chemistry , chromatography , inorganic chemistry , biochemistry , physics , organic chemistry , quantum mechanics
Ion and fluid transport through lens epithelial cells (LEC) is important for maintaining lens transparency. A water‐soluble chloride‐sensitive dye N‐ (ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide (MQAE) was used to measure the intracellular chloride concentration [Cl] i in a human LEC line (B3). Confluent B3 cells were incubated with MQAE in serum‐free medium and equilibrated in a high K + solution of varying extracellular chloride concentrations [Cl] o in the presence and absence of the ionophores tributyltin (TBT) and nigericin to exchange Cl − for OH − and K + for H + , respectively. Fluorescence emission, generated at 360 nm, was measured at 460 nm. Consistent with fluorescence quenching of MQAE by Cl, when MQAE‐loaded B3 cells were equilibrated in presence of the ionophores, MQAE fluorescence was maximum at 0 mM [Cl] o and exponentially decreased with increasing [Cl] o . Maximum MQAE fluorescence was observed in B3 cells loaded with 5 mM MQAE for 2 h and equilibrated in variable [Cl] o solutions with 5 μM nigericin and 10 μM TBT for 45 min. From the linear regression of a plot of the natural logarithm of the measured fluorescence as a function of [Cl] o , the baseline [Cl] i in B3 cells was calculated as 55 ± 1.6 mM. Hence at 163 mM [Cl] o , the calculated chloride equilibrium potential (E Cl ) was −28 mV which was lower than the reported E m value of − 33 mV for LEC. Therefore [Cl] i appears to be above equilibrium commensurate with inward Cl transport through a robust Na‐K‐2Cl cotransporter present in B3 cells (Lauf et al., Exp. Eye Res. 82 :–64, 2006). (Supported by WSU BMS Program).