一种用于测定刷状缘膜囊泡中阴离子交换活性的灵敏技术。大鼠肠上皮中对HCO3-和SITS具有不同亲和力的两种交换体的证据。

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium.

作者信息

Vaandrager A B, De Jonge H R

机构信息

Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, The Netherlands.

出版信息

Biochim Biophys Acta. 1988 Apr 7;939(2):305-14. doi: 10.1016/0005-2736(88)90075-2.

DOI:10.1016/0005-2736(88)90075-2

PMID:3355819

Abstract

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.

摘要

在平衡交换条件下，大鼠小肠刷状缘膜囊泡中高达70%的³⁶Cl⁻内流被发现是由SITS敏感的阴离子交换介导的。通过测定由大量Cl⁻梯度（内大于外）驱动的痕量¹²⁵I⁻摄取，该Cl⁻/阴离子交换的测量灵敏度可提高10 - 15倍，这种Cl⁻梯度是通过使囊泡通过阴离子交换柱产生的。用K⁺和缬氨霉素对囊泡膜进行电压钳制并不影响Cl⁻驱动的¹²⁵I⁻摄取，这表明“过冲”的I⁻摄取不是由电扩散电位介导的，而电扩散电位可能是由存在氯离子通道时的Cl⁻梯度产生的。通过研究各种阴离子对Cl⁻驱动的I⁻摄取的抑制作用，进一步对大鼠回肠和空肠刷状缘膜囊泡中的Cl⁻/阴离子交换进行了表征。2 mM的NO₃⁻、Cl⁻、SCN⁻和甲酸可抑制Cl⁻/I⁻交换达80%以上。回肠刷状缘膜囊泡在SO₄²⁻、SITS和HCO₃⁻对Cl⁻/I⁻交换的抑制作用方面表现出明显的异质性。大约30%的Cl⁻/I⁻交换对SO₄²⁻不敏感，对SITS的敏感性相对较低（IC₅₀ = 1 mM），但可被2 mM HCO₃抑制80%。推测该组分代表Cl⁻/OH⁻或Cl⁻/HCO₃交换。其余70%对SO₄²⁻（IC₅₀ = 0.5 mM）和SITS（IC₅₀ = 2.5 μM）高度敏感，但对HCO₃⁻不太敏感。这种交换活性的这一部分显示出与空肠囊泡中Cl⁻/I⁻交换非常相似的抑制特性。后者的过程也被2 mM草酸盐抑制80%。如本文所讨论的，两种交换体可能都参与了NaCl跨小肠绒毛细胞顶端膜的电中性转运。