ERK 1/2 is involved in the hypercarbia‐induced Na,K‐ATPase endocytosis

Objective Na, K‐ATPase activity of alveolar epithelial cells (AEC) plays a key role effecting alveolar fluid clearance (AFC). We observed that hypercarbia (60–120 mmHg) (HC) decreases AFC and Na,K‐ATPase activity by promoting its endocytosis from the plasma membrane. It has been described that activation of E xtracellular signal‐ R egulated K inase pathway (ERK) plays a role in endocytosis of the Na,K‐ATPase and so we set out to determine whether ERK is involved in the HC‐mediated downregulation of Na,K‐ATPase in AEC. Methods AEC were incubated in a Biospherix “C” Chamber in HC conditions and ERK activation was assessed using antibodies against phospho‐ERK and total‐ERK. The specificity of activation was confirmed by pre‐treating AEC with the ERK specific inhibitor, UO126, prior to HC. Na,K‐ATPase activity was assessed by 32 P‐ATP hydrolysis and α1‐subunit protein abundance at the plasma membrane was determined after cell surface biotinylation. Ras activation was determined by a Ras Activation assay kit. Results We found that ERK 1/2 was activated within 1–5 min and Ras was activated after 1 min of exposure to HC and this was prevented by U0126. In HC conditions, endocytosis of the Na,K‐ATPase was prevented when AEC were incubated with U0126. Summary High levels of CO 2 activate Ras and ERK 1/2 in AEC, which play a role in hypercarbic‐induced Na,K‐ATPase endocytosis. Funding : HL‐48129 and HL‐076139.