Blaug S, Hybiske K, Cohn J, Firestone G L, Machen T E, Miller S S
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Am J Physiol Cell Physiol. 2001 Aug;281(2):C633-48. doi: 10.1152/ajpcell.2001.281.2.C633.
Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na(+) channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance (R(T)) are -5.9 mV and 829 Omega x cm(2), respectively. The apical membrane potential (V(A)) is -40.7 mV, and the mean ratio of apical to basolateral membrane resistance (R(A)/R(B)) is 2.8. Apical amiloride hyperpolarized V(A) by 19.7 mV and tripled R(A)/R(B). A cAMP-elevating cocktail depolarized V(A) by 17.6 mV, decreased R(A)/R(B) by 60%, increased short-circuit current by 6 microA/cm(2), decreased R(T) by 155 Omega x cm(2), and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na(+) currents [linear current-voltage (I-V) relation] and forskolin-stimulated Cl(-) currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2--4 microl x cm(-2) x h(-1)). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na(+) transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl(-) transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na(+)] and [Cl(-)].
乳腺上皮31EG4细胞（MEC）以单层形式生长在滤器上，以分析有助于介导离子和液体跨上皮运输的顶端膜机制。逆转录聚合酶链反应（RT-PCR）显示存在囊性纤维化跨膜传导调节因子（CFTR）和上皮钠通道（ENaC）的信息，免疫显微镜检查显示两种蛋白均有顶端膜染色。CFTR也定位于天然人乳腺导管上皮的顶端膜。在对照条件下，跨上皮电位（顶端侧为负）和电阻（R(T)）的平均值分别为-5.9 mV和829Ω×cm²。顶端膜电位（V(A)）为-40.7 mV，顶端与基底外侧膜电阻的平均比值（R(A)/R(B)）为2.8。顶端应用氨氯地平使V(A)超极化19.7 mV，并使R(A)/R(B)增加两倍。一种升高环磷酸腺苷（cAMP）的混合剂使V(A)去极化17.6 mV，使R(A)/R(B)降低60%，使短路电流增加6 μA/cm²，使R(T)降低155Ω×cm²，并在很大程度上消除了对氨氯地平的反应。全细胞膜片钳测量显示氨氯地平抑制钠电流[线性电流-电压（I-V）关系]和福斯可林刺激氯电流（线性I-V关系）。一种电容探针法显示，在对照状态下，MEC单层要么吸收要么分泌液体（2-4 μl×cm⁻²×h⁻¹）。通过激活CFTR（cAMP）或阻断ENaC（氨氯地平）刺激液体分泌。这些数据加上等效电路分析表明：1）跨MEC的液体吸收由通过顶端膜ENaC的钠转运介导，液体分泌部分由通过顶端CFTR的氯转运介导；2）在这两种情况下，适当的抗衡离子通过紧密连接移动以维持电中性；3）CFTR、ENaC和紧密连接之间的相互作用使MEC能够吸收或分泌液体，并且在原位可能有助于控制管腔[Na⁺]和[Cl⁻]。
