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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI.

We investigated the regulatory transport processes that maintain intracellular K+ homeostasis in cultured rat glomerular mesangial cells (MCs). Intracellular K+ concentration ([K+]i) of quiescent MCs, passages 3-8, grown to subconfluence on glass cover slips, was assessed by spectrofluorometry using the K(+)-sensitive dye, K(+)-binding benzofuran isophthalate (PBFI). Serum-starved MCs were incubated at 37 degrees C in 5 microM PBFI for 90 min. Excitation ratios of luminescences at 340 and 380 nm, measured at a constant emission at 500 nm, were used to determine [K+]i. Ionophores valinomycin and nigericin were used to clamp [K+]i to known [K+]o and thereby obtain an intracellular calibration of dye. Dependence of fluorescence ratio on [K+]i conformed to Michaelis-Menten behavior, with a Km of 113 mM (n = 40). PBFI retains its sensitivity to alterations in [K+]i with pH change (pHi from 6.5 to 7.5) but is relatively insensitive when intracellular Na+ is greater than 75 mM and cell osmolarity exceeds 500 mM. Normal resting [K+]i for all experiments was determined in MCs to be 102 +/- 7 mM (n = 81) in a HCO3(-)-free HEPES-buffered solution. When MCs were exposed to ouabain, [K+]i fell to 48 +/- 6 mM and did not recover, suggesting presence of Na(+)-K(+)-ATPase. When MCs were exposed to furosemide, [K+]i transiently declined to 58 +/- 11 mM, which was followed by a rapid recovery to near steady state, indicating additional presence of Na(+)-K(+)-Cl- cotransporter. Recovery was completely abolished when MCs were exposed to ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)[1]


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