Regulation of Endothelial Intracellular Glutathione by ICAM‐1

Abstract

Recent work from our lab has uncovered a role for ICAM-1 in regulating endothelial glutathione levels. Here we provide further evidence for the involvement of this adhesion molecule in this process. Using cross-linking antibodies, total GSH was measured after ICAM-1 ligation on confluent endothelium. Next, confluent cells were treated overnight with 1, 10, or 100 ng/mL soluble ICAM-1 (sICAM-1) followed by total GSH measurements. Lastly, confluent endothelial cells were treated overnight with 10, 25, or 50 μM of a cell permeable peptide containing the transmembrane domain and cytoplasmic tail of ICAM-1, and then GSH was measured. Cross linking ICAM-1 on the endothelial surface, thus mimicking leukocyte adhesion, induced a significant increase in GSH levels at 16 hrs (135.02±15.32 nmol/mg protein vs 74.99±2.75 nmol mg/protein for untreated). Soluble ICAM-1 at 1 ng/mL also significantly elevated endothelial GSH levels (88.52±9.76 nmol/mg protein for 1 ng/mL sICAM-1 vs. 56.88±3.79 nmol/mg protein for untreated), and treatment with 25 and 50 μM of a cell permeable C-terminal fragment of ICAM-1 significantly increased GSH levels compared to the control peptide (36.14±1.07 and 39.65±1.27 nmol/mg protein for 25 and 50 μM ICAM-1 peptide, respectively, vs. 19.82±1.15 and 24.39±0.63 nmol/mg protein for 25 and 50 μM control peptide, respectively). These data provide novel evidence that ICAM-1 regulates endothelial cell redox status through modulation of pathways controlling intracellular glutathione.