Question:

Does signalling via PKA / SGK1 - Nedd4/2 control pulmonary Na⁺ transport by regulating the surface abundance of a-,b- and g-ENaC?

Methods:

Surface abundance of a-,b- and g-ENaC in H441 human airway epithelial cells was quantified by western analysis of surface exposed proteins that were isolated by biotinylation / streptavidin binding. PKA / SGK1 activity and the phosphorylation / abundance of endogenous Nedd4-2 were quantified by immuno-precipitation / Western analysis, whilst ENaC activity was assessed by quantifying whole cell / transepithelial current.

Result:

Selectively activating PKA by brief (20 min) exposure to cAMP-elevating drugs increased the abundance Ser²²¹ -phosphorylated, Ser³²⁷-phosphorylated and total Nedd4-2 but had no effect upon the abundance of Thr²⁴⁶-phosphorylatyed protein. Activating PKA also increased the surface abundance a-ENaC with no effect upon b- or g-ENaC. Selectively activating SGK1 (0.2 µM dexamethasone, 3 h) increased the abundance of Ser²²¹-, Ser³²⁷- and Thr²⁴⁶-phosphorylated Nedd4-2 and caused an SGK1-dependent increases in the surface abundance of a-, b- and g-ENaC. Neither stimulus had a major effect upon ENaC activity. Chronic (24 h) exposure to dexamethasone (0.2 µM), on the other hand, selectively increased the surface abundance of a-ENaC, induced Nedd4-2 -Thr²⁴⁶-phosphorylation and caused SGK1-dependent activation of ENaC. Activating PKA (20 min) under these conditions increased the abundance of Ser²²¹-, Ser³²⁷-, Thr²⁴⁶-phosphorylated and total Nedd4-2, augmented the amount of a-,b- and g-ENaC at the cell surface, and augmented ENaC activity.

Conclusion:

As well as evoking ENaC activity, chronic glucocorticoids stimulation allows this transport process to be controlled by cAMP / PKA.