采用短路电流法对大鼠附睾尾培养细胞的跨上皮氯离子转运进行研究。

Studies of transepithelial Cl- transport in cultured cauda epididymal cells of rats by the short-circuit current method.

作者信息

Leung A Y, Wong P Y

机构信息

Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories.

出版信息

J Physiol. 1992 Nov;457:391-406. doi: 10.1113/jphysiol.1992.sp019384.

DOI:10.1113/jphysiol.1992.sp019384

PMID:1297839

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1175737/

Abstract

Monolayer cultures of cauda epididymides from male Sprague-Dawley rats (210-230 g) were studied by the short-circuit current (ISC) technique to characterize the properties of the transepithelial chloride transport. In HCO(3-)-free, HEPES-buffered solution, adrenaline (0.23 microM) added to the basolateral side led to an increase in ISC and transepithelial conductance (gt). 2. Decreasing apical chloride concentration ([Cl-]a) progressively from 126.7 to 0 mM by substituting chloride with gluconate increased the ISC response to adrenaline (delta ISC) in a linear fashion with a slope of -1.6 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. Pretreating the tissue with a chloride channel blocker diphenylamine-2-carboxylate (DPC) on the apical side significantly reduced the slope to -4.9 x 10(-4) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. 3. By substituting apical chloride with various anions and measuring the change in ISC upon adrenaline stimulation, the selectivity sequence of the apical anion conductance was found to be NO3- approximately Br- > Cl- > I- > gluconate > isethionate. 4. When the monolayers were bathed with Krebs-Henseleit solution containing 25 mM HCO3- and 5% CO2, the delta ISC at each [Cl-]a as well as the dependence of delta ISC on [Cl-]a (slope = -3.3 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a) were significantly greater than the HCO(3-)-free counterpart. Addition of 0.1 mM acetazolamide or 0.5 mM SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to the basolateral side significantly reduced the effects of HCO3- and CO2. 5. When the tissues were bathed on both sides with HCO(3-)-free, HEPES-buffered solution and were clamped at various transepithelial potential differences (PDt) from +30 mV (lumen positive) to -30 mV (lumen negative), the relationship between the clamping current response to adrenaline (delta ICL) and the PDt applied was linear. Zero clamping current response was found at -6 mV. Decreasing [Cl-]a to 0 mM reduced the dependence of delta ICL on PDt and delta ICL was positive at all PDt tested. The response of the transepithelial conductance to adrenaline (delta gt) did not depend on the PDt applied but was reduced with decreasing apical chloride concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

摘要

采用短路电流（ISC）技术研究了雄性Sprague-Dawley大鼠（210 - 230克）附睾尾部的单层培养物，以表征跨上皮氯转运的特性。在不含HCO₃⁻的HEPES缓冲溶液中，添加到基底外侧的肾上腺素（0.23微摩尔）导致ISC和跨上皮电导（gt）增加。2. 通过用葡萄糖酸盐替代氯离子，使顶端氯离子浓度（[Cl⁻]a）从126.7毫摩尔逐渐降低至0毫摩尔，以线性方式增加了对肾上腺素的ISC反应（δISC），斜率为每毫摩尔[Cl⁻]a变化 -1.6×10⁻³微当量·小时⁻¹·厘米⁻²。在顶端用氯离子通道阻滞剂二苯胺 - 2 - 羧酸盐（DPC）预处理组织，显著将斜率降低至每毫摩尔[Cl⁻]a变化 -4.9×10⁻⁴微当量·小时⁻¹·厘米⁻²。3. 通过用各种阴离子替代顶端氯离子并测量肾上腺素刺激后的ISC变化，发现顶端阴离子电导的选择性顺序为NO₃⁻≈Br⁻＞Cl⁻＞I⁻＞葡萄糖酸盐＞羟乙磺酸盐。4. 当单层培养物用含有25毫摩尔HCO₃⁻和5% CO₂的Krebs-Henseleit溶液孵育时，每个[Cl⁻]a下的δISC以及δISC对[Cl⁻]a的依赖性（斜率 = -3.3×10⁻³微当量·小时⁻¹·厘米⁻²每毫摩尔[Cl⁻]a变化）显著大于不含HCO₃⁻的对应物。在基底外侧添加0.1毫摩尔乙酰唑胺或0.5毫摩尔SITS（4 - 乙酰氨基 - 4'-异硫氰酸基芪 - 2,2'-二磺酸）显著降低了HCO₃⁻和CO₂的作用。5. 当组织两侧用不含HCO₃⁻的HEPES缓冲溶液孵育，并在从 +30毫伏（管腔为正）到 -30毫伏（管腔为负）的各种跨上皮电位差（PDt）下钳制时，钳制电流对肾上腺素的反应（δICL）与施加的PDt之间的关系是线性的。在 -6毫伏时发现钳制电流反应为零。将[Cl⁻]a降低至0毫摩尔降低了δICL对PDt的依赖性，并且在所有测试的PDt下δICL均为正。跨上皮电导对肾上腺素的反应（δgt）不依赖于施加的PDt，但随着顶端氯离子浓度的降低而降低。（摘要截断于400字）