Cohn J A
Am J Physiol. 1987 Sep;253(3 Pt 1):G420-4. doi: 10.1152/ajpgi.1987.253.3.G420.
The T84 colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using 32Pi. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in 32P incorporation into four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasoactive intestinal peptide and suggests that T84 cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.
利用T84结肠上皮细胞系研究神经体液刺激离子转运过程中的蛋白质磷酸化。将生长在胶原包被滤膜上的T84细胞单层置于尤斯灌流小室中，以测量血管活性肠肽刺激的离子转运。刺激8 - 10分钟后出现活性分泌的最大刺激。使用二维凝胶电泳检测伴随刺激分泌的蛋白质磷酸化事件，以分离先前用32Pi标记的单层中的磷蛋白。在暴露于血管活性肠肽的8分钟内，检测到几种磷酸化事件，包括四种表观分子量分别为17,000、18,000、23,000和37,000的可溶性蛋白质中32P掺入量增加了2至5倍。在由二丁酰腺苷3',5'-环磷酸（cAMP）刺激的单层中也出现相同的磷酸化反应，表明cAMP介导这些细胞内事件。本研究表明，蛋白质磷酸化的变化伴随着血管活性肠肽的分泌作用，并提示T84细胞为研究此类磷酸化事件调节肠上皮细胞离子转运的可能性提供了一个有用的模型。
