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.

In alveolar epithelial cells, G-protein coupled-receptors agonists (GPCR) induce the recruitment of the Na,K-ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K-ATPase to occur, dephosphorylation of its alpha1-subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho-antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K-ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K-ATPase alpha1-subunit is an in vitro substrate for PP2A; and 3) glutathione S-transferase (GST)-fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K-ATPase alpha1-subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K-ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K-ATPase alpha1-subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.

Rationale: Endothelin-1 (ET-1) is increased in patients with highaltitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR). Objectives: To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation. Methods: Isolated perfused rat lung, transgenic rats deficient in ET B receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86 Rb 1 uptake. Measurements and Main Results: The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ET B , but not ET A . Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ET B receptors in the pulmonary vasculature (ET-B 2/2 ) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor L-NAME, but not by a guanylate cyclase inhibitor. Conclusions: We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ET B receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.

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.

PURPOSE:To determine whether exposure of the alveolar epithelium to airborne particles alters alveolar barrier function and decreases tight junction proteins. METHODS:Rat alveolar type II cells (ATII) were cultured on transwells inserts, and lung adenocarcinoma A549 on 100 mm culture dishes. These cells were exposed to particulate matter < 10 microns in diameter (PM10) (1,5 ,10 and 20 μg/cm 2 ) or vehicle for up to 24 hours, after which the plasma membranes were isolated and examined using immunofluorescence and immunoblotting to measure changes in occludin. Measurement of paracellular permeability to 4 kDa FITC-dextran and transepithelial electrical resistance (TER) were performed to assess barrier function. Mice were anesthetized and a 20-gauge angiocath was placed transorally into the trachea under direct visualization. We instilled either PM (200 μg) suspended in 50 μl of sterile PBS or 50 μl of sterile PBS in two equal aliquots, 3 min apart. Lungs were taken 24 hours later.RESULTS: PM10 at 10 and 20 μg/cm 2 caused a significant TER drop after 24 hours, changes occurring first at 3 hours with 20 μg/cm 2 (p<0.001). ATII paracellular permeability was increased (70%) after 24 hours of exposure to PM10 (20 μg/cm 2 ). The total abundance at the plasma membrane of occludin was decreased in alveolar epithelial cells after exposure to PM10 for 3 hours and in whole lungs of mice exposed PM10 for 24 hours. Also, there was a loss of interaction between the tight junction protein ZO-1 and occludin after exposure to PM10 for 3 hours. CONCLUSION:Particulate matter alters the alveolar epithelial barrier, possibly via a decrease in the tight junction protein occludin.CLINICAL IMPLICATIONS: Higher morbidity and mortality from pulmonary and cardiovascular events due to air pollution may be explained by an alveolar epithelial barrier dysfunction causing airborne particles to directly traverse the lung epithelium or indirectly induce pulmonary inflammation.