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.
Hg(2+) 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 Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) 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 Hg(2+)-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 Hg(2+) 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 Hg(2+)-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.
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524, USA.
Jitesh D
JD
Kawedia
Mengmeng
M
Jiang
Amit
A
Kulkarni
Holly E
HE
Waechter
Karl S
KS
Matlin
Giovanni M
GM
Pauletti
Anil G
AG
Menon
D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008628:Mercury / Q000633:toxicity*; D062793:Occludin; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D012469:Salivary Glands / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013363:Submandibular Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*
D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases; D008628:Mercury