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Mechanism of Tumor Necrosis Factor alpha (TNFα) Inhibition of B 0 AT1 Mediated Glutamine Transport in Rat Intestinal Epithelial Cell (IEC‐6)
Author(s) -
Talukder Jamil,
Bayakly Nabil,
Dawney Tonickee
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1068.20
Subject(s) - angiotensin ii receptor type 1 , chemistry , tumor necrosis factor alpha , microbiology and biotechnology , genistein , western blot , angiotensin ii , biochemistry , endocrinology , biology , receptor , gene
Background TNFα inhibits B 0 AT1‐mediated glutamine (Gln) transport by decreasing the affinity of the transporter for Gln on the brush border membrane (BBM) of IEC‐6 cells. TNFα has been shown to exert its effect on several proteins through different signaling pathways. Aim To identify the specific signaling pathway that TNFα activates to inhibit B 0 AT1‐mediated Gln transport in IEC‐6 cells. Methods IEC‐6 cells grown on 6‐well plates were treated with TNFα and/or specific inhibitors: Suramin, RpMBcAMP, Calphostin C, BAPTA, and Genistein for PKG, PKA, PKC, Ca 2+ chelator, and ERK/JUNK, respectively for 24 h. B 0 AT1 function was determined by measuring [ 3 H]‐Gln uptake. Cytosolic Ca 2+ was measured using Flu 3AM. RTQ‐PCR and Western blot analyses were performed. Results TNFα decreased B 0 AT1 activity in IEC‐6 cells (7.1±0.1 nmol/mg protein/2 min in control and 2.9±0.1 in TNFα treated cells, n=4, p<0.01). Only Genistein fully blocked TNFα mediated inhibition of B 0 AT1 activity (6.9±0.1 nmol/mg protein/2 min). Kinetic studies demonstrated that the protective mechanism of TNFα mediated inhibition of B 0 AT1 by Genistein was due to the restoration of affinity of B 0 AT1 for Gln. Molecular studies showed that both B 0 AT1 mRNA and B 0 AT1 protein levels on BBM remained unaltered in all groups. Conclusions These studies revealed that TNFα inhibits B 0 AT1 through ERK/JUNK pathway most likely by phosphorylation in IEC‐6 cells.