Fluid reabsorption in various epithelia is regulated by Na+ transport via apically located epithelial sodium channels (ENaC). Submandibular gland epithelial (SMG-C6) cells cultured in the absence of hydrocortisone or cAMP demonstrate diminished a-ENaC expression and Na+ transport properties similar to fetal distal lung epithelial cells. Increases in the abundance of the serum and glucocorticoid-regulated protein kinase 1 (sgk1), a phosphatidylinositol 3-kinase (PI3K) activated protein, can suppress ENaC degradation. Since treatment of SMG-C6 cells with dibutyryl cAMP (DbcAMP) for 24 hours increases a-ENaC expression and function, we hypothesized that the up-regulation of a-ENaC expression and amiloride-sensitive Na+ transport by DbcAMP involves an increase in sgk1 via the protein kinase A (PKA) and PI3K pathways. SMG-C6 cells treated with DbcAMP (1 mM) increased both sgk1 RNA levels (peaking at 30 minutes) and protein expression (peaking after 4 h and remained elevated at 24 h). DbcAMP-stimulated sgk1 mRNA expression was decreased by actinomycin D and protein expression was decreased by PKA inhibitors (H-89 and KT5720). Both pharmacological inhibition of PI3K with LY294002 or transfection with dominant negative PI3K construct reduced stimulated sgk1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP-activated transcription factor), and, after 24 h, decreased a-ENaC protein levels and amiloride-sensitive Na+ transport. In addition, the combination of PKA inhibitors with dominant negative PI3K synergistically attenuated all amiloride-sensitive Na+ transport activity. DbcAMP induction of sgk1 acts through the PI3K and PKA pathways and sgk1 may potentiatea-ENaC.
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