Regulation of Na⁺/H⁺ Exchanger in Dendritic Cells by Akt1

Abstract

Background/aims: Dendritic cells (DCs), antigen-presenting cells critically important for primary immune response and establishment of immunological memory, are activated by bacterial lipopolysaccharides (LPS) resulting in stimulation of Na(+)/H(+) exchanger, ROS formation and migration. The effects are dependent on phosphoinositide 3 (PI3) kinase and paralleled by Akt phosphorylation. The present study explored the contribution of the Akt isoform Akt1.

Methods: Cytosolic pH (pH(i)) (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence), Na(+)/H(+) exchanger activity (Na(+) dependent realkalinization after an ammonium pulse), cell volume (forward scatter in FACS analysis), and ROS production (2',7'-dichlorodihydrofluorescein diacetate [DCFDA] fluorescence) were determined in DCs isolated from bone marrow of mice lacking functional Akt1/PKBα (akt1(-/-)) and their wild type littermates (akt1(+/+)).

Results: Forward scatter was lower in akt1(-/-) than in akt1(+/+) DCs, whereas pH(i), Na(+)/H(+) exchanger activity and ROS formation were less in untreated akt1(-/-) and akt1(+/+) DCs. Exposure of DCs to LPS was followed by increase of forward scatter and ROS formation to a similar extent in akt1(-/-) and in akt1(+/+) DCs. A 4 hours treatment with either LPS (1µg/ml) or tert-butylhydroperoxide (tBOOH, 5 µM) significantly stimulated Na(+)/H(+) exchanger activity in both genotypes, effects, however, significantly blunted in akt1(-/-) DCs.

Conclusion: The present observations demonstrate that Akt1 is required for the full stimulation of Na(+)/H(+) exchanger activity by LPS or oxidative stress in dendritic cells.