Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study.
Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study.
复制标题
培养犬气道上皮细胞中 Na-K-Cl 共转运的调节：[3H]布美他尼结合研究。
DOI：
10.1152/ajpcell.1990.259.4.c557
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Boucher,RC
中科院分区：
文献类型：
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作者：
Haas,M;Johnson,LG;Boucher,RC
我们检测了[3H]布美他尼与犬气管和支气管上皮分离膜的结合，以及与这些细胞的原代培养融合。来自气管和支气管的粗质膜以饱和方式结合[3H]布美他尼；与支气管膜（K1/2约为3.5 μ m， B（max）约为7.5 μ mol/mg）相比，气管膜具有更高的亲和力，但最大结合（K1/2约为0.7 μ m， Bmax约为2.5 pmol/mg）较低。在这两种情况下，当Na， K或Cl从介质中去除时，可饱和结合减少了65%以上。在原代培养中，当将[3H]布美他尼（1.0微米）加入到气管（1.20 +/- 0.10 pmol结合/mg总细胞蛋白）和支气管（1.79 +/- 0.52 pmol/mg）培养液中时，可饱和[3H]布美他尼结合（被30倍过量未标记的布美他尼抑制）发生；顶端[3H]布美他尼的结合最小。异丙肾上腺素(10(-5)M；基底外侧暴露)使饱和基底外侧[3H]布美他胺与气管培养物的结合增加约100%，支气管培养物增加约30%。当根尖Cl从134 mM减少到4 mM时，以及当添加100 mM蔗糖使根尖和底侧介质都高渗时，也可以看到类似的结合增强。在后两种情况下，异丙肾上腺素很少或没有产生额外的结合增加。我们的研究结果表明，在β -肾上腺素能刺激犬气道上皮净盐分泌过程中，通过Na-K-Cl共转运的基底外侧Cl内流的增加与基底外侧膜中功能性共转运蛋白数量的实际增加有关，而不仅仅是由于离子梯度的变化。然而，共转运位点的增加可能是继发于顶端Cl通道的初始刺激，从而导致细胞收缩。
We examined [3H]bumetanide binding to membranes isolated from canine tracheal and bronchial epithelia and to confluent primary cultures of these cells. Crude plasma membranes from trachea and bronchus bind [3H]bumetanide in a saturable manner; tracheal membranes have a higher affinity but lower maximal binding (K1/2 approximately equal to 0.7 microM; Bmax approximately equal to 2.5 pmol/mg protein) than do bronchial membranes (K1/2 approximately equal to 3.5 microM; B(max) approximately equal to 7.5 pmol/mg). In both cases, saturable binding is reduced by greater than 65% when either Na, K, or Cl is removed from the medium. In primary cultures, saturable [3H]bumetanide binding (inhibited by a 30-fold excess of unlabeled bumetanide) occurs when [3H]bumetanide (1.0 microM) is added to the solution bathing the basolateral side of tracheal (1.20 +/- 0.10 pmol bound/mg total cell protein) and bronchial (1.79 +/- 0.52 pmol/mg) cultures; minimal binding is seen with apical [3H]bumetanide. Isoproterenol (10(-5) M; basolateral exposure) produces approximately 100% increase in saturable basolateral [3H]bumetanide binding to tracheal cultures and approximately 30% increase in bronchial cultures. Similar augmentation of binding is seen when apical Cl is reduced from 134 to 4 mM and when both apical and basolateral media are made hypertonic by addition of 100 mM sucrose. Under these latter two conditions, isoproterenol produces little or no additional increase in binding. Our results indicate that the increase in basolateral Cl influx via Na-K-Cl cotransport that must occur during beta-adrenergic stimulation of net salt secretion in canine airway epithelia is related to an actual increase in the number of functioning cotransporters in the basolateral membrane and is not simply due to a change in ion gradients. The increase in cotransport sites, however, may be secondary to initial stimulation of apical Cl channels, with resultant cell shrinkage.