兔胆囊上皮细胞顶端膜中性Na(+)-Cl-偶联转运的性质：III. 膜囊泡转运分析

The nature of the neutral Na(+)-Cl- coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles.

作者信息

Meyer G, Bottà G, Rossetti C, Cremaschi D

机构信息

Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.

出版信息

J Membr Biol. 1990 Nov;118(2):107-20. doi: 10.1007/BF01868468.

DOI:10.1007/BF01868468

PMID:2266544

Abstract

In rabbit gallbladder epithelium, a Na+/H+, Cl-/HCO3- double exchange and a Na(+)-Cl- symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na+, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na+ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (delta pH) was imposed [( H+]in greater than [H+]out, PD = 0), the addition of Na-gluconate to the external solution caused delta pH dissipation at a rate that followed saturation kinetics. Amiloride (10(-4) M) reduced the delta pH dissipation rate. Taken together, these data indicate the presence of Na+ and H+ conductances in addition to an amiloride-sensitive, electroneutral Na+/H+ exchange. An inwardly directed [Cl-] gradient (PD = 0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl-/OH- exchange. When both [Na+] and [Cl-] gradients (outwardly directed, PD = 0) were present, fluorescence quenching reached a maximum 20-30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na+] gradient alone or the same [Na+] gradient with Cl- at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na+ and Cl- concentration gradients and hydrochlorothiazide (5 x 10(-4) M). The decrease was not influenced by an inhibitor of Cl-/OH- exchange (10(-4) M furosemide) or of Na(+)-K(+)-2Cl- symport (10(-5) M bumetanide). We conclude that a Na+/H+ exchange and a Na(+)-Cl- symport are present and act simultaneously. This suggests that in intact tissue the Na(+)-Cl- symport is also likely to work in parallel with the Na+/H+ exchange and does not represent an induced homeostatic reaction of the epithelium when Na+/H+ exchange is inhibited.

摘要

在兔胆囊上皮细胞中，存在Na⁺/H⁺、Cl⁻/HCO₃⁻双交换和Na⁺-Cl⁻同向转运，但对完整组织进行的实验无法确定这两种转运系统是否同时发挥作用。因此，制备了分离的顶端质膜囊泡。用Na⁺预加载后，注入无钠培养基会导致囊泡内稳定酸化（用吖啶橙荧光猝灭法监测），向外部溶液中添加Na⁺可逆转这种酸化。尽管程度较小，但在电势差（PD）等于0的实验中也会发生酸化。如果施加预设的pH差（ΔpH）（[H⁺]in大于[H⁺]out，PD = 0），向外部溶液中添加葡萄糖酸钠会导致ΔpH以遵循饱和动力学的速率消散。氨氯地平（10⁻⁴ M）降低了ΔpH消散速率。综合这些数据表明，除了对氨氯地平敏感的电中性Na⁺/H⁺交换外，还存在Na⁺和H⁺电导。内向的[Cl⁻]梯度（PD = 0）不会诱导囊泡内酸化。因此，在该制备物中，没有证据表明存在Cl⁻/OH⁻交换。当同时存在[Na⁺]和[Cl⁻]梯度（外向，PD = 0）时，荧光猝灭在囊泡注入后20 - 30秒达到最大值，然后迅速下降。在仅存在[Na⁺]梯度或两侧Cl⁻浓度相等的相同[Na⁺]梯度的情况下未观察到这种下降。同样，在同时存在Na⁺和Cl⁻浓度梯度以及氢氯噻嗪（5×10⁻⁴ M）的情况下，这种下降被消除。这种下降不受Cl⁻/OH⁻交换抑制剂（10⁻⁴ M 呋塞米）或Na⁺-K⁺-2Cl⁻同向转运抑制剂（10⁻⁵ M 布美他尼）的影响。我们得出结论，存在Na⁺/H⁺交换和Na⁺-Cl⁻同向转运且它们同时发挥作用。这表明在完整组织中，Na⁺-Cl⁻同向转运也可能与Na⁺/H⁺交换并行发挥作用，并且在Na⁺/H⁺交换受到抑制时不代表上皮细胞的诱导性稳态反应。