Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC.

Ambient particulate matter affects occludin distribution and increases alveolar transepithelial electrical conductance.

BACKGROUND AND OBJECTIVE: Inhaled particulate matter (PM) causes lung inflammation and epithelial dysfunction. However, the direct effect of PM on alveolar epithelial barrier integrity is not well understood. Our aim is to determine whether PM exposure affects the alveolar epithelial cells (AEC) transepithelial electrical conductance (Gt) and tight junction (TJ) proteins. METHODS: Human AEC (A549) and primary rat AEC were exposed to PM of <10 µm in size (PM(10) ) and diesel exhaust particles (DEP), using titanium dioxide (TiO(2) ) as a control for particle size effects. Gt and permeability to fluorescein isothiocyanate-dextran (FITC-Dextran) were measured to assess barrier integrity. TJ integrity was evaluated by analysing penetration of Lanthanum nitrate (La(3+) ) under transmission electron microscopy. Surface proteins were labelled with biotin and analysed by western blot. Immunofluorescence was performed to assess colocalization of TJ proteins including occludin and zonula occludens-1 (ZO-1). PM induced dissociation of occludin-ZO-1 was evaluated by co-immunoprecipitation. RESULTS: PM(10) and DEP increased Gt and disrupted TJ after 3 h of treatment. PM(10) and DEP induced occludin internalization from the plasma membrane into endosomal compartments and dissociation of occludin from ZO-1. Overexpression of antioxidant enzymes manganese superoxide dismutase (MnSOD) and catalase, prevented PM-induced Gt increase, occludin reduction from the plasma membrane and its dissociation from ZO-1. CONCLUSIONS: PM induces alveolar epithelial dysfunction in part via occludin reduction at the plasma membrane and ZO-1 dissociation in AEC. Furthermore, these effects are prevented by overexpression of two different antioxidant enzymes.

TSH regulates pendrin membrane abundance and enhances iodide efflux in thyroid cells.

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular (125)iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.