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-mu-M 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 K(m) of 113 mM (n = 40). PBFI retains its sensitivity to alterations in [K+]i with pH change (pH(i) 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. Exposure to Ba2+ led to an immediate increase in [K+]i to 124 +/- 8 mM followed by a rapid return to steady-state [K+]i. MCs exposed to tetraethylammonium exhibited a behavior similar to those exposed to Ba2+; resting [K+]i increased to 123 +/- 10 mM. Effects of angiotensin II (ANG II) and serotonin (5-HT) on Na+-K+-ATPase was assessed. None of these agents significantly altered resting [K+]i. ANG II and 5-HT stimulated Na+-K+-ATPase by 28 and 41%, respectively, as measured during recovery of [K+]i toward normal after a diuretic-induced fall in [K+]i. We conclude from these findings that 1) MCs possess ouabain-sensitive Na+-K+-ATPase, loop diuretic-sensitive Na+-K+-Cl- cotransporter, and barium-sensitive K+ channels; 2) ANG II and 5-HT stimulate Na+-K+-ATPase; and 3) a continuous determination of [K+]i and an examination of its regulatory mechanisms in MCs may be achieved through use of PBFI.