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

Abstract

Hg2ϩ 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 2ϩ affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg 2ϩ 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 2ϩ -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 2ϩ 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 2ϩ

Occludin is required for TRPV1-modulated paracellular permeability in the submandibular gland

“…In SMG-C6 cells, the initial TER value was 516.2698.62 V . cm 2 , which was consistent with previous reports in the same cell line (Kawedia et al, 2008) and represented a relatively 'tight' epithelium according to the description of electrical characteristics (Anderson and Van Itallie, 2009). 1 mmol/l capsaicin did not induce any change to TER values within 60 min (Fig.…”

“…2A, lower panels). Moreover, we extracted the cytoplasm and membrane protein fractions as previously described (Kawedia et al, 2008). Capsaicin induced a significant increase in occludin protein in the cytoplasm fraction within 60 min, together with a decrease in the membrane fraction.…”

Occludin: One Protein, Many Forms

“…Contrasting studies, however, again highlight the potential multiplicity of occludin pSer and pThr sites and related cell-specific signaling effectors that mediate the functional diversity of this protein. In this respect, elevated pSer/pThr occludin levels have been correlated to increased epithelial permeability (58,88,130) as well as to BBB dysfunction in encephalitic mouse and human brain (127). Collins et al (26) also showed a reduction in pSer/pThr occludin levels in BAECs in response to physiological cyclic strain (i.e., a barrier-strengthening influence).…”

Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: Implication of the PKA pathway

“…Several studies have demonstrated that PKA activity is important for the regulation and localization of TJ-associated proteins. Indeed, subcellular localization of claudin 1, paracellin-1 or occludin is dependent on phosphorylation by PKA in both physiological and pathological states (French et al, 2009;Ikari et al, 2006;Kawedia et al, 2008). Interestingly, Klingler et al (2000) showed that disruption of TJ barriers after calcium depletion could be preserved when PKA inhibitors were used.…”