Wills N K, Purcell R K, Clausen C
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550-2781.
J Membr Biol. 1992 Feb;125(3):273-85. doi: 10.1007/BF00236439.
Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (Isc) than A6 epithelia (44 +/- 2 and 27 +/- 2 microA/cm2, respectively). However, transepithelial conductances (GT) were similar for the two epithelia (0.62 +/- 0.04 mS/cm2 for 2F3 and 0.57 +/- 0.04 mS/cm2 for A6) because of reciprocal differences in cellular (Gc) and paracellular (Gj) conductances. Significantly lower Gj and higher Gc values were observed for 2F3 epithelia than A6 (Gj = 0.23 +/- 0.02 and 0.33 +/- 0.04 mS/cm2 and Gc = 0.39 +/- 0.16 and 0.26 +/- 0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (Ec) and the amount of transcellular Na+ current under open-circuit conditions (Ic) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the "dual-layer" and "access resistance" models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79 +/- 0.23 mS/cm2) than A6 epithelia (0.37 +/- 0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4 +/- 0.04 and 1.2 +/- 0.1 microF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48 +/- 1.67 and 2.95 +/- 0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions.
以往对完整上皮组织进行的阻抗分析研究因结缔组织或平滑肌的存在而变得复杂。我们现在报告该方法首次应用于培养的上皮单层细胞。阻抗分析被用作一种非破坏性方法，用于推导从肾细胞系A6及其亚克隆细胞系2F3生长而来的上皮组织的定量形态测量参数。选择亚克隆2F3细胞系与A6进行比较是因为其固有地具有更高的Na⁺转运速率。与先前结果一致，2F3上皮组织显示出比A6上皮组织显著更高的氨氯地平敏感短路电流（Isc）（分别为44±2和27±2 μA/cm²）。然而，由于细胞（Gc）和细胞旁（Gj）电导的相互差异，两种上皮组织的跨上皮电导（GT）相似（2F3为0.62±0.04 mS/cm²，A6为0.57±0.04 mS/cm²）。观察到2F3上皮组织的Gj显著低于A6，而Gc则高于A6（Gj分别为0.23±0.02和0.33±0.04 mS/cm²，Gc分别为0.39±0.16和0.26±0.10 mS/cm²）。尽管如此，两种上皮组织的细胞Na⁺转运驱动力（Ec）和开路条件下的跨细胞Na⁺电流（Ic）相似。推导了三种基于形态学的不同等效电路模型来评估上皮组织的阻抗特性：一种考虑侧向细胞间隙电阻的分布式模型和两种模型（“双层”和“接入电阻”模型），这两种模型校正了基底膜向下方滤膜支撑物的小的充满液体的突起的阻抗。尽管数据可以用分布式模型拟合，但顶膜与基底外侧膜电阻之比的估计值不合理地大。相比之下，其他模型提供了统计学上更优的拟合以及膜电阻比的合理估计。双层模型和接入电阻模型还提供了相似的顶膜和基底外侧膜电导及电容估计值。此外，两种模型都提供了关于基底外侧突起的电导和面积的新信息。2F3的顶膜电导估计值（0.79±0.23 mS/cm²）显著高于A6上皮组织（0.37±0.07 mS/cm²），但在顶膜电容（2F3和A6分别为1.4±0.04和1.2±0.1 μF/cm²）或基底外侧膜电导（3.48±1.67和2.95±0.40 mS/cm²）方面未检测到显著差异。两种上皮组织相似的基底外侧膜特性可能由它们在开路条件下相当的跨细胞Na⁺电流来解释。
