Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI
1Department of Medicine, Case Western Reserve University, Cleveland Veterans Affairs Medical Center, Ohio 44106.
Abstract:
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)
Insights
This study reveals that cultured rat mesangial cells utilize Na(+)-K(+)-ATPase and Na(+)-K(+)-Cl- cotransporter to maintain intracellular potassium homeostasis. These transporters are crucial for regulating potassium levels in these kidney cells.
Area of Science:
- Nephrology
- Cell Physiology
- Biochemistry
Background:
- Intracellular potassium homeostasis is vital for cellular function.
- Glomerular mesangial cells (MCs) play a key role in kidney function.
- Understanding ion transport in MCs is crucial for kidney health.
Purpose of the Study:
- To investigate the regulatory transport mechanisms maintaining intracellular potassium ([K+]i) in cultured rat MCs.
- To characterize the role of specific ion transporters in MCs.
- To establish a method for accurately measuring [K+]i in MCs.
Main Methods:
- Spectrofluorometry using the K(+)-sensitive dye PBFI to measure [K+]i.
- Calibration of the dye using ionophores (valinomycin and nigericin) to clamp [K+]i.
- Exposure of MCs to ouabain and furosemide to assess transporter function.
- HEPES-buffered solution for experiments.
Main Results:
- Resting [K+]i in MCs was determined to be 102 ± 7 mM.
- Ouabain treatment reduced [K+]i to 48 ± 6 mM, indicating Na(+)-K(+)-ATPase activity.
- Furosemide treatment caused a transient [K+]i decrease to 58 ± 11 mM, followed by recovery, suggesting Na(+)-K(+)-Cl- cotransporter presence.
- Furosemide-induced recovery was abolished by ouabain.
Conclusions:
- Cultured rat MCs possess both Na(+)-K(+)-ATPase and Na(+)-K(+)-Cl- cotransporter.
- These transporters are essential for maintaining intracellular potassium homeostasis in MCs.
- The study provides insights into the complex ion transport systems in kidney mesangial cells.
