Abstract: FR-PO210

Mineralocorticoid Receptor Antagonists Augment Arginine Transport and Nitric Oxide Generation through Modulation of Cationic Amino Acid Transporter-1 in Human Umbilical Vein Endothelial Cell

Session Information

Category: Hypertension

  • 1103 Vascular Biology and Dysfunction

Authors

  • Schwartz, Doron, Sourasky Medican Center, Tel Aviv, Israel
  • Shashar, Moshe, Sourasky Medican Center, Tel Aviv, Israel
  • Schwartz, Idit F., Tel Aviv Medical Sourasky Center, Tel Aviv, Israel
Background

.Blockade of the mineralocorticoid receptor (MCR) has been shown to improve endothelial function far beyond blood pressure control. In the current studies we looked at the effect of MCR antagonists on the activity of cationic amino acid transporter-1 (CAT-1), a major modulator of endothelial nitric oxide (NO) generation.

Methods

. Using radio-labeled arginine, {[3H] L-arginine} uptake was determined in Human umbilical vein endothelial cells (HUVEC) following incubation with either spironolactone or epleronone with or without silencing of the MCR. Western blotting for CAT-1, PKCα and their phosphorylated forms were performed. NO generation was measured by the Griess reaction .

Results

.Both Spironolactone and epleronone significantly increased endothelial arginine transport, an effect which was augmented by co-incubation with aldosterone and blunted by either silencing of the MCR or Co-administration of amyloride. Following MCR blockade but not amyloride, we identified two bands for CAT-1. The addition of tunicamycin (a de-glycosylation agent) or silencing of the MCR resulted in disappearance of the extra band and prevented the increase in arginine transport. Spironolactone but not epleronone decreased CAT-1 phosphorylation through inhibition of PKCα (CAT-1 inhibitor). Subsequently, the concentration of NO2/NO3 (stable NO metabolites) following incubation with both MCR antagonists significantly increased. This was attenuated by silencing of MCR or tunicamycin. GO 6076 (PKCα inhibitor) augmented the increase of NO metabolites only in the epleronone treated cells.

Conclusion

Spironolactone and epleronone augment arginine transport and NO generation through modulation of CAT-1 in endothelial cells. Both MCR antagonists activate CAT-1 by facilitating its glycosylation while only spironolactone inhibits PKCα.