Turner J R, Rill B K, Carlson S L, Carnes D, Kerner R, Mrsny R J, Madara J L
Department of Pathology, Brigham and Women's Hospital and the Harvard Digestive Disease Center, Boston, Massachusetts 02115, USA.
Am J Physiol. 1997 Oct;273(4):C1378-85. doi: 10.1152/ajpcell.1997.273.4.C1378.
Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 +/- 2% (P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 microM ML-7 or 40 microM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 (P < 0.01), which was inhibited by ML-9 (P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.
紧密连接是亲水性溶质跨肠上皮被动转运的限速屏障。钠 - 葡萄糖共转运激活后，肠紧密连接的通透性增加。由于此前对这种生理性紧密连接调节的分析仅限于完整黏膜，因此对该过程潜在机制的剖析受到限制。为了表征这一过程，我们构建了一个简化模型，该模型由转染了肠钠 - 葡萄糖共转运体SGLT1的Caco - 2肠上皮细胞组成。SGLT1转染细胞单层表现出生理性钠 - 葡萄糖共转运。SGLT1激活导致跨上皮电阻（TER）下降22±5%（P<0.001）。同样，添加根皮苷使SGLT1失活会使TER增加24±2%（P<0.001）。紧密连接通透性的增加具有大小选择性，小营养分子大小的分子（如甘露醇）通量增加，而大分子（如菊粉）通量未增加。SGLT1依赖性紧密连接通透性增加受到肌球蛋白轻链激酶抑制剂（20μM ML - 7或40μM ML - 9）的抑制，这表明肌球蛋白调节轻链（MLC）磷酸化参与紧密连接调节。MLC磷酸化分析显示，SGLT1激活后增加了2.08倍（P<0.01），这被ML - 9抑制（P<0.01）。因此，用葡萄糖和肌球蛋白轻链激酶抑制剂孵育的单层细胞与用根皮苷孵育的单层细胞相当。ML - 9也抑制小肠黏膜中SGLT1介导的紧密连接调节（P<0.01）。这些数据表明，上皮细胞是SGLT1激活后生理性紧密连接调节的介质。紧密连接调节与MLC磷酸化之间的密切关系表明，上皮紧密连接通透性调节的关键步骤可能是肌球蛋白ATP酶介导的连接周肌动球蛋白环收缩以及随后对紧密连接的物理张力。
