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 I SC 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 I SC. 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.
Highlights
During postnatal lung adaptation alveolar fluid has to be removed to promote air breathing
Since phosphorylation of AKT and serum- and glucocorticoid-regulated kinase 1 (SGK1) by mTORC2 is essential for their full activity, we investigated the effect of PP242 on insulin stimulation
We demonstrate that Phosphatidylinositide 3-kinases (PI3K) and AKT mediate the stimulatory action on ENaC-like amiloride-sensitive channels in a mTORC2dependent manner
Summary
During postnatal lung adaptation alveolar fluid has to be removed to promote air breathing. Na+ enters the cells through epithelial sodium channels (ENaC) in the apical plasma membrane and is actively extruded through the basolateral membrane by Na,K-ATPases Both transporters are essential for vectorial Na+ transport, the uptake of Na+ by ENaC is usually rate-limiting (O’Brodovich et al 1990; Hummler et al 1996). Impaired AFC leads to wet lung syndrome and respiratory distress syndrome (RDS; Helve et al 2004) In preterm infants, these morbidities occur more frequently and studies showed that smaller amounts of ENaC are expressed, suggesting a dependency of AFC on ENaC function (Helve et al 2004; Janer et al 2010). Lung edema in at-risk subjects may be prevented by increasing the function of ENaC or Na,K-ATPases, which could be beneficial for preterm infants
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