OPTICAL MEASUREMENTS OF INTRACELLULAR PH IN SINGLE LLC-PK1 CELLS - DEMONSTRATION OF CL-HCO3 EXCHANGE
OPTICAL MEASUREMENTS OF INTRACELLULAR PH IN SINGLE LLC-PK1 CELLS - DEMONSTRATION OF CL-HCO3 EXCHANGE
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DOI：
10.1073/pnas.83.2.522
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发表时间：
1986-01-01
影响因子：
11.1
通讯作者：
BORON, WF
中科院分区：
文献类型：
--
作者：
CHAILLET, JR;AMSLER, K;BORON, WF
使用光学方法连续监测单个培养的LLC-PK 1细胞中的细胞内pH（pHi）。通过将快速生长或静止的细胞暴露于染料的渗透前体，将附着在盖玻片上的细胞加载pH敏感染料4“，5”-二甲基-5（和-6）-羧基荧光素。从单个细胞中染料的细胞内吸收光谱计算pHi。对于在不含HCO 3的林格氏溶液中孵育的细胞，pHi从NH 4+预脉冲施加的酸负荷指数恢复。由于恢复是Na+依赖性和阿米洛利敏感性的，它可能是由质膜上的Na-H交换引起的。在HCO 3-林格氏溶液中，外部Cl-去除导致pHi可逆地增加约1.5倍。0.3.通过50 μ M 4，4”-二异硫氰基二苯乙烯-2，2”-二磺酸盐（DIDS）或通过在名义上不存在HCO 3-的情况下进行Cl-去除，这种pHi增加大幅降低。在恒定的pCO 2下将[HCO 3-]o从25 mM降低至5 mM（将pHo从7.4降低至6.7）导致pHi可逆地下降约0.2.通过DIDS、通过去除细胞外Cl-或通过在标称不存在HCO 3-的情况下进行相同的pHo偏移，这种pHi变化大大减小。改变[Cl-]o或pHo引起的pHi变化不受Na+去除的抑制。我们的数据表明，LLC-PK 1细胞具有钠非依赖性氯-HCO 3交换，这种转运蛋白可能是重要的钠-氢交换器在确定pH值。
An optical method was used to continuously monitor intracellular pH (pHi) in single cultured LLC-PK1 cells. Rapidly growing or quiescent cells, attached to coverslips, were loaded with the pH-sensitive dye 4'',5''-dimethyl-5(and -6)-carboxyfluorescein by exposing them to the dye''s permeant precursor. pHi was calculated from the intracellular absorbance spectrum of the dye in a single cell. For cells incubated in HCO3--free Ringer''s solution, pHi recovered exponentially from acid loads applied by NH4+ prepulsing. Because the recovery was Na+-dependent and amiloride-sensitive, it was probably caused by Na-H exchange at the plasma membrane. In HCO3- Ringer''s solution, external Cl- removal caused pHi to reversibly increase by .apprxeq. 0.3. This pHi increase was substantially reduced by 50 .mu.M 4,4''-diisothiocyanostilbene-2,2''-disulfonate (DIDS) or by conducting the Cl- removal in the nominal absence of HCO3-. Reducing [HCO3-]o from 25 to 5 mM at constant pCO2 (lowering pHo from 7.4 to 6.7) caused pHi to reversibly fall by .apprxeq. 0.2. This pHi change was greatly diminished by DIDS, by removing of extracellular Cl-, or by performing the same pHo shift in the nominal absence of HCO3-. The pHi changes induced by altering [Cl-]o or pHo were not inhibited by Na+ removal. Our data indicate that LLC-PK1 cells possess a Na-independent Cl-HCO3 exchanger and that this transporter may be as important as the Na-H exchanger in determining pHi.