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Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3Kdependent pathway. In these cells, dopamine and [beta]-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase [[alpha].sub.1]-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway. thyroid hormone

Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-KATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) ceils. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell fine, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic pll0 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-KATPase [[alpha].sub.1] protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway. triiodothyronine

Na-K-ATPase protein is critical for maintaining cellular ion gradients and volume and for transepithelial ion transport in kidney and lung. Thyroid hormone, 3,3',5-triiodo-L-thyronine ([T.sub.3]), given for 2 days to adult rats, increases alveolar fluid resorption by 65%, but the mechanism is undefined. We tested the hypothesis that [T.sub.3] stimulates Na-K-ATPase in adult rat alveolar epithelial cells (AEC), including primary rat alveolar type II (ATII) cells, and determined mechanisms of the [T.sub.3] effect on the Na-KATPase enzyme using two adult rat AEC cell lines (MP48 and RLE-6TN). [T.sub.3] at [10.sup.-8] and [10.sup.-5] M increased significantly hydrolytic activity of Na-K-ATPase in primary ATII cells and both AEC cell lines. The increased activity was dose dependent in the cell lines ([10.sup.-9]-[10.sup.-4] M) and was detected within 30 min and peaked at 6 h. Maximal increases in Na-K-ATPase activity were twofold in MP48 and RLE-6TN cells at pharmacological [T.sub.3] of [10.sup.-5] and [10.sup.-4] M, respectively, but increases were statistically significant at physiological [T.sub.3] as low as [10.sup.-9] M. This effect was [T.sub.3] specific, because reverse T3 (3,3',5'-triiodo-L-thyronine) at [10.sup.-9]-[10.sub.-4] M had no effect. The [T.sub.3]-induced increase in Na-K-ATPase hydrolytic activity was not blocked by actinomycin D. No significant change in mRNA and total cell protein levels of Na-K-ATPase were detected with [10.sup.-9]-[10.sup.-5] M [T.sub.3] at 6 h. However, [T.sub.3] increased cell surface expression of Na-K-ATPase [[alpha].sub.1]- or [[beta].sub.1]-Subunit proteins by 1.7- and 2-fold, respectively, and increases in Na-K-ATPase activity and cell surface expression were abolished by brefeldin A. These data indicate that [T.sub.3] specifically stimulates Na-K-ATPase activity in adult rat AEC. The up-regulation involves translocation of Na-K-ATPase to plasma membrane, not increased gene transcription. These results suggest a novel nontranscriptional mechanism for regulation of Na-K-ATPase by thyroid hormone. alveolar cell lines; protein translocation; sodium pump

[Na.sup.+]-[K.sup.+]-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase [Na.sup.+]-[K.sup.+]-ATPase activity and mRNA levels. We sought to define the mechanism of [Na.sup.+]-[K.sup.+]-ATPase gene upregulation by GC. In a rat alveolar epithelial cell line (RLE), dexamethasone (Dex) increased [[beta].sup.1]-subunit [Na.sup.+]-[K.sup.+]-ATPase mRNA expression two- to threefold within 3 h after exposure to the GC. The increased gene expression was due to increased transcription as demonstrated by nuclear run-on assays, whereas mRNA stability remained unchanged. Transient transfection of 5' deletion mutants of a [[beta].sub.1] promoter-reporter construct demonstrated a 1.5- to 2.2-fold increase in promoter activity by Dex. All of the 5' deletion constructs contained partial or palindromic GC regulatory elements (GRE) and responded to GC. The increased expression of promoter reporter was inhibited by RU-486, a GC receptor (GR) antagonist, suggesting the involvement of GR. The palindromic GRE at -631 demonstrated Dex induction in a heterologous promoter construct. Gel mobility shift assays using RLE nuclear extracts demonstrated specific binding to this site and the presence of GR. We conclude that GC directly stimulate transcription of [Na.sup.+]-[K.sup.+]-ATPase [[beta].sub.1] gene expression in adult rat lung epithelial cells through a GR-dependent mechanism that can act at multiple sites. sodium pump; glucocorticoid receptor; glucocorticoid response element; promoter; alveolar fluid; ion transport; type II cells

Whole cell perforated patchclamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was -60 mV, and depolarizing voltage steps activated voltage-gated [K.sup.+] (Ky) channels. The voltage-activated currents exhibited a mean reversal potential of -32 reV. Complete activation was achieved at -10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in [K.sup.+] selectivity, suggesting contributions of multiple Kv [alpha]-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between -30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv [alpha]-subunits (Kv1.1, Kv1.3, Kv 1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three [beta]-subunits (Kv[beta]l.1, Kv[beta]2.1, and Kv[beta]3.1), and two [K.sup.+] channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv [alpha]-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv [alpha]-, [beta]-, and KChIP subunits explains the variability in inactivation gating and [K.sup.+] selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal [K.sup.+] secretion across the alveolar epithelium. voltage-gated potassium channels; potassium ion secretion; oxygen-sensitive potassium channels; alveolar fluid clearance

The Na+ channel and Na+-K+-ATPase are essential for the perinatal removal of alveolar solute and fluid. It was hypothesized that maternal glucocorticoids (GCs) increased Na+-K+-ATPase gene expression in a fetal lung epithelial cell line because Na+-pump mRNA and activity increase before birth. The results showed that GCs increased Na+-K+-ATPase transcription in a fetal lung epithelial cell line. The findings suggest important therapeutic implications, one of which is optimizing treatment of premature infants by maternal GCs.

Madin-Darby canine kidney (MDCK) cells have enhanced production of the sodium-potassium antiporter mRNA upon exposure to hyperoxic regimes. The mechanism of gene regulation in MDCK is investigated using promoter transfection techniques and deletion mutants of the promoter sequence. Results reveal that a 61-base pair segment located between nucleotide residues 102 and 41 is responsible for the induction of Na-K-ATPase during hyperoxia.

Research shows that acute hyperoxia in rats modifies sodium pump activity, affecting alveolar sodium resorption and active sodium transport. Experiments designed to assess the effects of hypoxia on lung sodium-potassium-adenosine triphosphatase activity using healthy male Sprague-Dawley rats are described. Isolated, perfused lung preparations are prepared.

Research shows that type II alveolar epithelial cell migration on provision matrix proteins is crucial to the reepithelialization of denuded alveoli in lung repair. Type I alveolar epithelial cells (AEC) migrate by haptotaxis more readily via substrate-bound fibronectin than they do on type I collagen. Alpha2Beta1-integrin mediates AEC migration on type I collagen.

The effects of maternally administered dexamethasone on fetal lung Na-K-adenosinetriphosphatase (ATPase) expression were analyzed in pregnant Sprague-Dawley rats. Maternal dexamethasone treatment increased the steady-state levels of Na-K-ATPase mRNA in developing fetal rat lungs in a time-dependent manner. Furthermore, the glucocorticoid differentially regulated the beta1 and alpha1 subunits of the sodium pump in developing fetal lungs.

The biochemical role of Na-K-adenosine triphosphatase (ATPase) and Na+ pump expression were analyzed in alveolar type II (ATII) cells isolated from the lungs of adult rats after acute exposure to oxygen. Chronic hyperoxic injury in rat ATII cells did not alter normal Na-K-ATPase ion transport. However, hyperoxia-induced acute lung injury decreased maximal enzyme activity and levels of alpha1-subunit antigenic protein.

The adherence of type II alveolar cells to fibronectin, fibrinogen, vitronectin and fibrin is mediated by integrins during reepithelialization,following acute lung injury in rats. Arginine-glycine-aspartic acid peptide suppresses the adhesion of type II cells to fibronectin, fibrinogen and fibrin. The alpha(sub v)beta(sub 3)-integrin partially mediates adhesion to vitronectin. Adhesion of the type II cells to fibronectin, fibrinogen and fibrin is useful for reepithelialization of the alveolus.

The developing fetal lung acquires the ability to exchange gases, water and salt by producing Na,K-ATPase. The protein components for Na,K-ATPase are already present in the fetal rat lung at fetal day 17, but the rest of the subunits for functional expression of Na,K-ATPase are only produced a few days before the neonatal day. The administration of ouabain established that these lungs were already functional as soon as the rats are born.

Hyperoxia increases the expression of gamma-glutamyl transpeptidase (GGT) mRNA, glutathione disulfide, and the protein-bound glutathione (S-glutathiolated protein) in neonatal rat lungs. The levels of GGT protein and enzyme activity are higher in hyperoxia-exposed rats than in rats grown in room air. The levels fall during the recovery after exposure to room air for three days. S-glutathiolation appears to be a mechanism for the protection of lungs against oxidative injury, and a regulatory modification of protein sulfhydryl groups.

In the study of localizing the enzyme GGT within rat alveolar epithelial in vivo and to identify its expression by culture type II cells as they disintegrate into type I cells showed that the greater density of GGT per cell antibody binding in situ is in type I cells than type II cells. This results indicates that high level of immunoreactive with low specific activity and high molecular-weight are the characteristic of type I cells. This factor is supported by the in vitro increasing amounts of this immunoreactive protein in cultured type II cells.

Electrophoretic DNA size analysis and flow cytometric analysis of the influence of type II alveolar epithelial cells on the apoptosis of mesenchymal cells reveals that serum-free conditioned media (CM) from primary type II cell cultures of rat inhibit tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis and modulate the mesenchymal cell responses to apoptosis-induced repair and cell damage. CM causes a reduction in the percentage of TNF-alpha-induced DNA damaged cells. CM factor is sensitive to trypsin and binds with Mono-Q anion-exchange resin to display a discrete peak. Heat-stable peptides from the alveolar cells mediate the protective effect of CM, which is concentration dependent.