Thyroid hormone stimulates Na-K-ATPase activity and its plasma membrane insertion in rat alveolar epithelial cells

Abstract

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 (T3), given for 2 days to adult rats, increases alveolar fluid resorption by 65%, but the mechanism is undefined. We tested the hypothesis that T3 stimulates Na-K-ATPase in adult rat alveolar epithelial cells (AEC), including primary rat alveolar type II (ATII) cells, and determined mechanisms of the T3 effect on the Na-KATPase enzyme using two adult rat AEC cell lines (MP48 and RLE-6TN). T3 at 10-8 and 10-5 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 (10-9-10-4 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 T3 of 10-5 and 10-4 M, respectively, but increases were statistically significant at physiological T3 as low as 10-9 M. This effect was T3 specific, because reverse T3 (3,3',5'-triiodo-l-thyronine) at 10-9-10-4 M had no effect. The T3-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 10-9-10-5 M T3 at 6 h. However, T3 increased cell surface expression of Na-K-ATPase alpha1- or beta1-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 T3 specifically stimulates Na-K-ATPase activity in adult rat AEC. The upregulation 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.

3,3′,5-Triiodo-l-thyronine Up-regulation of Na,K-ATPase Activity and Cell Surface Expression in Alveolar Epithelial Cells Is Src Kinase- and Phosphoinositide 3-Kinase-dependent

“…T3 did not significantly alter the amount of total cell PKB/Akt protein in MP48 cells; however, T3 stimulated the level of phosphorylation of Ser 473 of PKB/Akt in a dose-dependent manner from 10 Ϫ8 to 10 Ϫ5 M (Fig. 3A), consistent with the dose response effects of T3 on Na,K-ATPase activity (12). T3-induced PKB/Akt phosphorylation at Ser 473 was detectable from 10 min to 12 h of T3 stimulation (Fig.…”

“…3A) and stimulated Na,K-ATPase activity in a dose-dependent manner (12). The parallel increases in Na,K-ATPase and PI3K activity induced by T3 supported the likelihood that T3 induced Na,K-ATPase activity through PI3K.…”

“…In our previous study (12), T3 enhanced the cell surface expression of both the Na,KATPase ␣ 1 and ␤ 1 subunits, whereas the total cell amount of those subunit proteins was not altered, but the T3-induced increase in Na,K-ATPase ␣ 1 protein at plasma membrane was sensitive to a protein-trafficking inhibitor, brefeldin A, that disrupts the protein trafficking from Golgi complex to plasma membrane. Those observations indicated that T3-induced cell surface expression of Na,K-ATPase may be derived from translocation of this enzyme.…”

“…Na,K-ATPase Activity Assay-The hydrolytic activity of Na,KATPase was measured as ouabain-sensitive ATP hydrolysis under V max conditions by measuring the release of P i from ATP as previously described (12). Na,K-ATPase specific activity was calculated as the difference in P i concentration/mg of protein/min in the absence and presence of 10 mM ouabain.…”

“…Thyroid hormone differentially regulates Na,K-ATPase isoforms in both tissue-and cell type-specific manners (1). Recently, we reported that in AECs T3 also stimulates rapidly the enzyme activity and cell surface expression of Na,KATPase in a transcription-independent manner (12); however, the signaling transduction pathway(s) is not defined. Identifying the signal transduction molecule(s) involved in T3 nontranscriptional stimulation of Na,K-ATPase would provide insight into the molecular mechanism(s) of thyroid hormone function.…”