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Leukotriene D4 Requires PKC α- Akt Signaling Pathway to Inhibit Na^+- Dependent Alanine Cotransporter (ASCT1) in Enterocytes

Jamilur R. Talukder, Jaleesa Wright, Antara Jaima, Deja McIntosh

Background: Varieties of inflammatory cytokines are produced during chronic enteritis including inflammatory bowel disease (IBD), that leads to malabsorption of nutrients and diarrhea. The inflammatory mediators are produced from different tissues of the body. Arachidonic acid metabolite derived via Lipoxygenase pathway, leukotriene D4 (LT) inhibits Na^+-dependent alanine (Ala) cotransport (ASCT1, solute carrier, slc1a4) in the apical membrane of enterocytes by decreasing the affinity of cotransporter. However, the intracellular mechanism of LT-mediated inhibition of ASCT1 activity is unknown.

Objective:To investigate the intracellular mechanism of leukotriene D4 (LT) mediated inhibition of ASCT1 activity in enterocytes.

Methods: Rat intestinal epithelial cells (IEC-6) were grown on transwell plates. [3H]-Ala uptake was measured in 10 days postconfluent cells using a scintillation counter. IEC-6 cells were treated with different inhibitors in 8 days postconfluent to intercept different checkpoints of pathways for LT-mediated inhibition of ASCT1 activity. Intracellular Ca²⁺ and cAMP levels were measured. Immunoblotting, qRT-PCR, and immunocytochemistry were performed following the standard protocols.

Results: LT treatment increased more than 2.5-fold [(cAMP)i] and [(Ca2+ )i]. PKA, PKC-δ and -θ inhibitor did not reverse the LT-mediated inhibition of ASCT1 activity. However, PKC-α inhibitor antagonized LT effect on ASCT1 activity. Further downstream of PKC-α pathway, tyrosine kinase (Akt) inhibitor also reversed LT-mediated inhibition of ASCT1 activity. Immunoblotting, qRT-PCR, immunocytochemistry, and Kinetics studies demonstrated that the mechanism of decreased affinity of ASCT1 by LT was due to decrease in affinity of ASCT1 for Ala transport that was restored by Akt inhibitor.

Conclusion: Therefore, we conclude that LT inhibits ASCT1 activity by decreasing the affinity of ASCT1 to Ala through Ca2+-dependent PKCα-Akt pathway in enterocytes.