Morris A P, Cunningham S A, Benos D J, Frizzell R A
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
J Biol Chem. 1992 Mar 15;267(8):5575-83.
The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist, stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization but was reduced when the cells formed tight junctions.
导致囊性纤维化（CF）的基因最近已被克隆和测序。当该基因转染到CF上皮细胞中时，其正常转录本可纠正CF的潜在缺陷，即恢复cAMP依赖性Cl⁻分泌。因此，该基因编码的蛋白质，命名为“囊性纤维化跨膜传导调节因子”（CFTR），以某种方式参与Cl⁻分泌反应。在本文中，我们将人结肠腺癌细胞系HT - 29的未极化（亲本）和极化（Cl.19A）克隆中CFTR基因表达与cAMP和Ca²⁺依赖性Cl⁻分泌进行了关联研究。发现这些细胞系在传代后4天表达同样高水平的CFTR mRNA。此外，蛋白质表达（通过免疫沉淀测定）也相同。生成cAMP的激动剂福斯可林对未极化细胞的¹²⁵I流出几乎没有影响。相反，该激动剂使极化细胞中的¹²⁵I流出增加了3倍。未极化细胞中缺乏反应并非由于福斯可林无法提高cAMP水平。神经降压素，一种Ca²⁺动员激动剂，刺激了两种细胞系的¹²⁵I流出。在极化细胞中，接种后8天这种反应的幅度减弱。此时，未分化的细胞系获得了一定的cAMP反应性。后一种效应与多细胞层区域内单层的出现平行。从极化细胞顶端膜片进行的细胞贴附式膜片钳记录显示存在福斯可林刺激的8 - pS Cl⁻通道；在福斯可林刺激的未极化细胞中未观察到通道活性。在两种细胞系中均发现了Ca²⁺激活的Cl⁻通道。与¹²⁵I流出数据一致，极化细胞系中对[Ca²⁺]i的单通道激活反应较小。从这些研究中，我们可以得出结论，在mRNA和蛋白质水平上测量的CFTR表达与结肠细胞对生成cAMP的激动剂作出反应分泌氯离子的能力不相关。cAMP依赖性Cl⁻分泌需要细胞极化；具体而言，需要界定顶端膜。分化过程中CFTR细胞定位的差异可能解释了我们的结果。相反，Ca²⁺刺激的Cl⁻分泌独立于细胞极化发生，但当细胞形成紧密连接时会减少。
