PHYSIOLOGICAL REGULATION OF EPITHELIAL TIGHT JUNCTIONS IS ASSOCIATED WITH MYOSIN LIGHT-CHAIN PHOSPHORYLATION

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 junct ions is increased. Because previous analyses of this physiological tig ht junction regulation have been restricted to intact mucosae, dissect ion of the mechanisms underlying this process has been limited. To cha racterize this process, we have developed a reductionist model consist ing of Caco-2 intestinal epithelial cells transfected with the intesti nal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectant s demonstrate physiological Nai-glucose cotransport. Activation of SGL T1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin i ncreases TER by 24 +/- 2% (P < 0.001). The increased tight junction pe rmeability is size selective, with increased flux of small nutrient-si zed molecules, e.g., mannitol, but not of larger molecules, e.g., inul in. SGLT1-dependent increases in tight junction permeability are inhib ited by myosin light-chain kinase inhibitors (20 mu M ML-7 or 40 mu M ML-9), suggesting that myosin regulatory Light-chain (MLC) phosphoryla tion is involved in tight junction regulation. Analysis of MLC phospho rylation 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-medi ated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of phys iological tight junction regulation subsequent to SGLT1 activation. Th e intimate relationship between tight junction regulation and MLC phos phorylation 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.