REGULATION OF INTRACELLULAR POTASSIUM IN MESANGIAL CELLS - A FLUORESCENCE ANALYSIS USING THE DYE, PBFI
REGULATION OF INTRACELLULAR POTASSIUM IN MESANGIAL CELLS - A FLUORESCENCE ANALYSIS USING THE DYE, PBFI
复制标题
DOI：
10.1152/ajprenal.1992.262.3.f462
复制
发表时间：
1992-03-01
影响因子：
--
通讯作者：
GANZ, MB
中科院分区：
文献类型：
--
作者：
KASNER, SE;GANZ, MB
我们研究了在培养的大鼠肾小球系膜细胞（MCs）中维持细胞内K+稳态的调节性转运过程。 通过荧光分光光度法，使用K+敏感染料，K+结合苯并呋喃三乙酸酯（PBFI），评估在盖玻片上生长至亚汇合的静止MCs（第3-8代）的细胞内K+浓度（[K+]i）。 将血清饥饿的MCs在37 ℃下在5 μ M PBFI中孵育90 min。使用在500 nm处恒定发射下测量的340和380 nm处的发光激发比来确定[K+]i。 离子载体缬氨霉素和尼日利亚菌素用于将[K+]i夹持到已知的[K+]o，从而获得染料的细胞内校准。 荧光比对[K+]i的依赖性符合Michaelis-Menten行为，K（m）为113 mM（n = 40）。 PBFI保持其对[K+]i随pH变化（pH（i）从6.5至7.5）的敏感性，但当细胞内Na+大于75 mM且细胞渗透压超过500 mM时相对不敏感。在无HCO 3的HEPES缓冲溶液中，所有实验的正常静息[K+]i在MCs中测定为102 +/- 7 mM（n = 81）。 当MCs暴露于哇巴因时，[K+]i下降至48 +/- 6 mM并且没有恢复，表明Na+-K+-ATP酶的存在。 当MCs暴露于呋塞米时，[K+]i短暂下降至58 +/- 11 mM，随后迅速恢复至接近稳态，表明Na+-K+-Cl-共转运蛋白的额外存在。 当MCs暴露于哇巴因时，恢复完全消失。 暴露于Ba 2+导致[K+]i立即增加至124 +/- 8 mM，随后快速恢复至稳态[K+]i。 暴露于四乙基铵的MC表现出与暴露于Ba 2+的MC相似的行为;静息[K+]i增加至123 +/- 10 mM。评估了血管紧张素II（ANG II）和5-羟色胺（5-HT）对Na+-K+-ATP酶的影响。 这些药物均未显著改变静息[K+]i。 ANG II和5-HT分别刺激Na+-K+-ATP酶28%和41%，这是在利尿剂诱导的[K +]i下降后[K +]i恢复正常期间测量的。 结果提示：MCs具有哇巴因敏感的Na ~+-K ~+-ATP酶、袢利尿剂敏感的Na ~+-K ~+-Cl ~-协同转运体和钡敏感的K ~+通道; ANG Ⅱ和5-HT可刺激Na ~+-K ~+-ATP酶; PBFI可连续测定MCs的[K ~+]i并探讨其调节机制。
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.