Abstract
Purpose :
Our lab previously reported elevated levels of citrulline and arginine in plasma samples of patients with Type II diabetes compared to diabetic controls. We found that citrulline and arginine together induced angiogenic features in human retinal endothelial cells (HRMEC) and increased expression of total and phosphorylated eNOS and NO production. In this study, we sought to determine the signaling pathway by which citrulline and arginine activate eNOS and their effect on retinal cell permeability.
Methods :
HRMEC were treated with 30 uM citrulline and 70 uM arginine (cit + arg) for 2 hours, and cell lysate was collected for Western blot. Blots were probed for phosphorylated and total expression of the following eNOS-activating molecules: Akt, AMPK, PKA, PKC, and CaMKII. HRMEC were treated with cit + arg, and eNOS localization was determined via immunofluorescence. HRMEC were treated with cit + arg on Transwell inserts for 24 hours, and cell permeability was measured using FITC-Dextran and trans-endothelial electrical resistance (TEER). HRMEC were treated with cit + arg for 24 hours, and VE-Cadherin, beta-catenin, claudin-5, and Zo-1 protein expression and localization were determined via Western blot and immunofluorescence.
Results :
AMPK (p = 0.025) and Akt (p = 0.050) displayed significantly increased phosphorylated protein expression in HRMEC treated with cit + arg, but total AMPK (p = 0.70) and total Akt (p = 0.20) were not altered. Cit + arg increased the ratio of phosphorylated to total AMPK (p = 0.039), indicating activation of AMPK, but there was no change in the pAKt/Akt ratio (p = 0.12). HRMEC treated with cit + arg showed loss of membrane and cytoplasm localization of eNOS compared to untreated cells. Cit + arg led to increased FITC-Dextran (p = 1.5 x 10-5) and decreased TEER (p = 0.0018) compared to untreated controls, indicating an increase in cell permeability. Immunofluorescence of membrane proteins revealed a loss of claudin-5 localization to the membrane. Western blot analysis showed no changes in expression of the membrane proteins.
Conclusions :
These data demonstrate that citrulline and arginine activate AMPK, relocate eNOS, and increase cell permeability in retinal endothelial cells. This suggests that citrulline and arginine activate eNOS via AMPK to produce NO which increases permeability of the cells through loss of claudin-5 localization to the membrane.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.