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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

The Protein Kinase A Pathway Contributes to Hg²⁺-Induced Alterations in Phosphorylation and Subcellular Distribution of Occludin Associated with Increased Tight Junction Permeability of Salivary Epithelial Cell Monolayers

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio (J.D.K., M.J., A.G.M.); Laboratory of Epithelial Pathobiology, Department of Surgery, University of Chicago, Chicago, Illinois (H.E.W., K.S.M.); and Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, Ohio (A.K., G.M.P.)

Hg²⁺ is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg²⁺ affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg²⁺ decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg²⁺-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg²⁺ treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg²⁺-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren's syndrome, and possibly other disorders of fluid transport.

Address correspondence to: Dr. Anil G. Menon, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. E-mail: anil.menon{at}uc.edu