We investigated whether a poly-L-arginine (poly-L-Arg) -induced increase in the tight-junction permeability of FITC-labeled dextran (MW 4.4 kDa, FD-4) is associated with a transient increase in transepithelial electroresistance (PD) and short-circuit current (Isc) immediately after poly-L-Arg exposure and occurs following tight-junction disassembly-related cellular signaling in the rabbit nasal epithelium.
A transient PD and Isc increase was associated with increased Cl
- secretion induced by poly-L-Arg but not with the enhanced paracellular FD-4 permeability. Enhanced paracellular FD-4 permeability induced by poly-L-Arg was not changed by treatment with inhibitors of possible Ca
2+ mobilization pathways followed by poly-L-Arg exposure, suggesting that the promotional effect of poly-L-Arg is independent of Ca
2+-related signaling.
Protein kinase C (PKC) and tyrosine phosphatase inhibitors suppress an increase in tight-junction permeability by poly-L-Arg, indicating that serine/threonine may have been phosphorylated via Ca
2+-independent PKC and tyrosine may have dephosphorylated the junction protein. ZO-1, a tight-junction-associated protein, and occludin, an integral membrane protein localized at the tight junction, were monitored immuno-fluorescently after preincubation with PKC and tyrosine phosphatase inhibitors followed by poly-L-Arg treatment, showing that ZO-1 and occludin were internalized via serin/threonine phosphorylation by PKC activation and via tyrosine dephosphorylation, providing tight-junction disassembly.
We concluded that poly-L-Arg enhances the paracellular permeability of macromolecular drugs via serin/threonine phosphorylation of ZO-1 involving Ca
2+-independent PKC activation and tyrosine dephosphorylation of occludin in the rabbit nasal epithelium. Such phosphorylation and dephosphorylation disperses junction proteins, particularly ZO-1 and occludin, into cytoplasm, possibly followed by tight-junction disassembly. These findings should prove useful in developing transnasal delivery systems for macromolecular drugs with polycationic materials as solute transport enhancers.
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