NA+-H+ EXCHANGE IN GASTRIC GLANDS AS MEASURED WITH A CYTOPLASMIC-TRAPPED, FLUORESCENT PH INDICATOR
NA+-H+ EXCHANGE IN GASTRIC GLANDS AS MEASURED WITH A CYTOPLASMIC-TRAPPED, FLUORESCENT PH INDICATOR
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DOI：
10.1073/pnas.81.23.7436
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
MACHEN, TE
中科院分区：
文献类型：
--
作者：
PARADISO, AM;TSIEN, RY;MACHEN, TE
采用ph敏感、荧光、细胞质捕获染料2”，7”-双（羧乙基）-5(6)-羧基荧光素（BCECF）检测高压灌注和胶原酶消化法分离兔胃腺中Na+-H+交换。通过渗透细胞膜并将细胞外溶液滴定到不同的pH值，以细胞质pH （pHc）来校准装载bcecf的腺体的荧光。在一组实验中，在无Cl溶液中，腺体用0.1 mM瓦巴因处理45分钟，以提高细胞胞质摩尔Na+浓度（[Na+]c）到高水平。随后将这些细胞悬浮在无Na+的林格溶液中（生成[Na+]c [Na+] 0 [Na+] 0），使细胞迅速酸化（t1/2[半衰期]）。60 s) from pHc .apprxeq.7.15到pHc .apprxeq。6.55. 随后加入100 mM Na+或Li+，但不加入K+，使细胞的pHc迅速增加（t1/2 .apprxeq）。30 s)向控制值移动。当用1mm阿米洛利预平衡瓦苦因处理的腺体10分钟后，ph值的变化被阻断，并通过添加10 .mu来克服这种阻断。莫能菌素（一种人工将Na+交换为H+的离子载体）。在另一组实验中，在含Cl的林格氏溶液中，用30mm NH4Cl处理4分钟，然后将NH4Cl从溶液中洗涤，从而使腺体酸化。在此条件下，ph值从7.02下降到0.0.0.eq。6.5;碱化后的细胞恢复到对照ph值，刺激Na+ (t1/2 .apprxeq)。60 s)，但不受K+的影响，并被1 mM阿米洛利抑制。进一步添加10亩也克服了这一障碍。莫能菌素。胃腺含有一种Na+- h +交换剂，它独立于Cl-，不被K+激活，被1mm阿米洛利阻断。这种交换剂可能位于腺体细胞的浆膜上。在高腔酸性环境下，Na+-H+交换可能在氧合细胞和主细胞的ph调节中起重要作用，在这种情况下，H+可能会快速向细胞内扩散。
The pH-sensitive, fluorescent, cytoplasmic-trapped dye 2'',7''-bis(carboxyethyl)-5(6)-carboxyfluorecein (BCECF) was used to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution to different pH vlaues. In one set of experiments in Cl--free solutions, glands were treated with 0.1 mM ouabain for 45 min to increase cellular cytosolic molar Na+ concentration ([Na+]c) to high levels. Subsequent suspension of these cells in a Na+-free Ringer''s solution (to generate [Na+]c > [Na+]o) caused cells to acidify rapidly (t1/2 [half-time] .apprxeq. 60 s) from pHc .apprxeq. 7.15 to pHc .apprxeq. 6.55. Subsequent addition of 100 mM Na+ or Li+, but not K+, caused cells rapidly to increase pHc (t1/2 .apprxeq. 30 s) toward the control value. These changes of pHc were blocked when ouabain-treated glands had been preequilibrated for 10 min with 1 mM amiloride, and this block was overcome by adding 10 .mu.M monensin (an ionophore that artificially exchanges Na+ for H+). In another set of experiments in Cl--containing Ringer''s solution, glands were acid-loaded by treatment with 30 mM NH4Cl for 4 min, followed by washing the NH4Cl from the solutions. Under these conditions, pHc decreased from 7.02 to .apprxeq. 6.5; subsequent alkalinization of cells back to control pHc was stimulated the Na+ (t1/2 .apprxeq. 60 s), but not K+, and was inhibited by 1 mM amiloride. This amiloride block also was overcome by further addition of 10 .mu.M monensin. Gastric glands contain a Na+-H+ exchanger that appears independent of Cl-, not activated by K+ and blocked by 1 mM amiloride. This exchanger is likely localized to the serosal membrane of gland cells. Na+-H+ exchange may play an important role in regulation of pHc in oxyntic and chief cells exposed to high luminal acidity, where back diffusion of H+ into cells may occur at rapid rates.