Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells.
Abstract Alveolar fluid clearance is driven by vectorial Na(+) transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na(+) transport was studied in isolated alveolar cells from 18-19-day gestational age rat fetuses. Equivalent short-circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose-dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na(+) channel) activity in experiments with permeabilized apical or basolateral membrane. 5-(N-Ethyl-N-isopropyl)amiloride inhibition of ISC was not affected, however, benzamil-sensitive ISC was increased in insulin-stimulated monolayers. The application of LY-294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF-1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF-1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na(+) transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.
Division of Neonatology, Center for Pediatric Research Leipzig, Hospital for Children & Adolescents, University of Leipzig, Leipzig, 04103, Germany.
Charlott
C
Mattes
Mandy
M
Laube
Ulrich H
UH
Thome
AKT; SGK1; alveolar cells; epithelial Na+ channel (ENaC); insulin