Berglund J J, Riegler M, Zolotarevsky Y, Wenzl E, Turner J R
Department of Pathology, Wayne State University, Detroit, Michigan 48201, USA.
Am J Physiol Gastrointest Liver Physiol. 2001 Dec;281(6):G1487-93. doi: 10.1152/ajpgi.2001.281.6.G1487.
Na(+)- nutrient cotransport-dependent regulation of paracellular permeability has been demonstrated in rodent intestine and human intestinal epithelial cell lines. In cell lines this regulation is associated with phosphorylation of myosin II regulatory light chain (MLC). However, the subcellular localization of phosphorylated MLC during this regulation has not been studied and regulation of paracellular permeability and MLC phosphorylation has not been studied in isolated human intestine. To evaluate these events in human jejunum, isolated mucosa was mounted in Ussing chambers, characterized electrophysiologically, and then immunostained using anti-phosphorylated MLC and anti-total MLC antisera. MLC phosphorylation was assessed by calculating the ratio of anti-phosphorylated MLC signal to anti-total MLC signal within defined regions. Transmucosal resistance of mucosae without active Na(+)-glucose cotransport was 37 +/- 3% greater than that of mucosae with active Na(+)-glucose cotransport within 15 min. Quantitative double-label immunofluorescence showed that the phosphorylated MLC-to-total MLC ratio increased by 45 +/- 4% within the perijunctional actomyosin ring when Na(+)-glucose cotransport was active. Thus regulation of transmucosal resistance by Na(+)-glucose cotransport is accompanied by increased MLC phosphorylation within the perijunctional actomyosin ring. These data support the proposed critical role of the perijunctional cytoskeleton in physiological regulation of human small intestinal paracellular permeability.
在啮齿动物肠道和人肠上皮细胞系中，已证实钠（Na⁺）-营养物共转运依赖性对细胞旁通透性的调节作用。在细胞系中，这种调节与肌球蛋白II调节轻链（MLC）的磷酸化有关。然而，在此调节过程中磷酸化MLC的亚细胞定位尚未得到研究，且在分离的人肠道中尚未研究细胞旁通透性和MLC磷酸化的调节。为了评估人空肠中的这些事件，将分离的黏膜安装在尤斯灌流小室中，进行电生理特性分析，然后用抗磷酸化MLC和抗总MLC抗血清进行免疫染色。通过计算在定义区域内抗磷酸化MLC信号与抗总MLC信号的比值来评估MLC磷酸化。在15分钟内，无活性Na⁺-葡萄糖共转运的黏膜的跨黏膜电阻比有活性Na⁺-葡萄糖共转运的黏膜高37±3%。定量双标记免疫荧光显示，当Na⁺-葡萄糖共转运活跃时，在连接周肌动球蛋白环内，磷酸化MLC与总MLC的比值增加了45±4%。因此，Na⁺-葡萄糖共转运对跨黏膜电阻的调节伴随着连接周肌动球蛋白环内MLC磷酸化的增加。这些数据支持了连接周细胞骨架在人小肠细胞旁通透性生理调节中所起关键作用的观点。
