Leukotriene D4 dephosphorylates ASCT1 through Ca2+ dependent PKCα‐Akt pathway in intestinal epithelial cells (1095.11)

Abstract

Arachidonic acid metabolite derived via Lipoxygenase pathway, leukotriene D4 (LT) inhibits Na+‐dependent alanine cotransport (ASCT1, solute carrier, SLC1A4) in intestinal epithelial cell brush border membrane (BBM) by decreasing the affinity of cotransporter. However, the intracellular mechanism of LT mediated inhibition of ASCT1 is unknown. This study was designed to investigate the intracellular mechanism of ASCT1 inhibition by LT in enterocytes. [3H]‐Ala uptake was measured in 10 days postconfluent rat intestinal epithelial cells (IEC‐6) grown on transwell plates. IEC‐6 cells were treated with different inhibitors intercepting different checkpoints of pathways for LT mediated inhibition of ASCT1 at 8 days postconfluence. LT treatment decreased ASCT1 activity and increased more than 2.5‐fold intracellular cAMP. However, protein kinase A (PKA)inhibitor did not reverse the LT mediated inhibition of ASCT1. LT increased 2‐fold cytosolic Ca2+ and PKC‐α antagonized LT effect on ASCT1 activity. In contrast, PKC‐δ and ‐θ inhibitor did not reverse LT mediated inhibition of ASCT1. Further downstream of PKC‐α pathway, tyrosine kinase (Akt) inhibitor also reversed LT mediated inhibition of ASCT1 activity. Kinetics, western blotting, and qRT‐PCR studies demonstrated that the mechanism of reversal of LT mediated inhibition of ASCT1 by Akt inhibitor was due to the restoration of affinity of the cotransporter. Immunocytochemical and dephosphorylation studies revealed that ASCT1 was dephosphorylated by LT treatment. Therefore, we conclude that LT inhibits ASCT1 activity by dephosphorylation through Ca2+‐dependent PKCα‐Akt pathway in enterocytes.Grant Funding Source: NSF