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)
Kasner
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SE
Department of Medicine, Case Western Reserve University, Cleveland Veterans Affairs Medical Center, Ohio 44106.
Ganz
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MB
D000818:Animals; D001464:Barium / Q000494:pharmacology; D001572:Benzofurans; D002478:Cells, Cultured; D004976:Ethacrynic Acid / Q000494:pharmacology; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D005665:Furosemide / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis; D007700:Kinetics; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence
D001572:Benzofurans; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; C073886:potassium-binding benzofuran isophthalate; D001464:Barium; D010042:Ouabain; D005665:Furosemide; D004976:Ethacrynic Acid; D011188:Potassium