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Fig. 2. tMLCK induction reduces barrier function. (A) TER decreases progressively after tMLCK induction. TER, normalized to control monolayers handled in parallel, fell progressively after doxycycline washout. TER 8-18 hours after tMLCK induction was similar to that seen as much as 48 hours after doxycycline washout (data not shown). Data shown are means ± s.e.m. of triplicate samples and are representative of more than ten independent experiments. Similar results were obtained with five independently generated clones. (B) Inhibition of myosin light chain kinase by addition of 10 µM ML-7 to control monolayers (closed bars) and monolayers expressing tMLCK (open bars) causes TER of the latter to increase to that of control monolayers not expressing tMLCK. tMLCK expression was induced for 18 hours and ML-7 added for 1 hour before TER measurement. Data shown are means ± s.e.m. of triplicate samples and are representative of four independent experiments. (C) tMLCK expression increases transepithelial flux of the small paracellular tracer mannitol, but not inulin, demonstrating size-selective increases in paracellular permeability as a consequence of tMLCK-induced MLC phosphorylation. Data shown are means ± s.e.m. of triplicate samples and are representative of three independent experiments.
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