Abstract 19862: HDL Stimulates Endothelial CAT-1 Expression and L-Arginine Uptake: A Novel Mechanism Leading to Endothelial-Protective Effects of HDL That is Profoundly Altered in Patients With Coronary Disease
Background: Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) exerts important anti-atherogenic effects. High-density lipoprotein from healthy subjects (HDLHS) stimulates endothelial NO production; however, the underlying mechanisms remain to be further defined. In the present study we therefore characterized the effects of HDLHS and HDL from patients with coronary disease (HDLCAD) on cationic amino acid transporter-1 (CAT-1), the main endothelial L-arginine transporter, and endothelial uptake of L-arginine, the substrate of eNOS.
Methods: HDLHS (n=15) and HDLCAD (n=20) was isolated by sequential ultracentrifugation. The effects of HDL on endothelial mRNA and protein expression of CAT-1 were determined, and cellular localization of CAT-1 was examined by confocal microscopy. NO production was measured by electron spin resonance spectroscopy. Endothelial L-arginine uptake was determined after [3H]-labeling of L-arginine. The role of CAT-1 for HDL-dependent endothelial L-arginine uptake and NO production was determined after CAT-1 inhibition by siRNA knockdown or by N-ethylmaleimide (NEM) or lysine.
Results: HDLHS substantially increased endothelial CAT-1 expression, both on mRNA and protein level (+82.1 and +74.8% vs. PBS, P<0.01). Moreover, HDLHS induced endothelial membrane translocation of CAT-1 and increased caveolar association of CAT-1 and eNOS. HDLHS stimulated total and CAT-1 mediated endothelial L-arginine uptake. Importantly, inhibition of CAT-1 activity by siRNA knockdown or by NEM and lysine profoundly reduced the effect of HDLHS on endothelial NO production. In marked contrast, HDLCAD did not stimulate endothelial expression and membrane translocation of CAT-1 and rather inhibited endothelial L-arginine uptake.
Conclusion: The present study demonstrates for the first time that HDLHS stimulates endothelial expression and membrane translocation of CAT-1, resulting in increased L-arginine uptake. Of note, stimulation of CAT-1 by HDLHS was critical for the capacity of HDL to stimulate endothelial NO production. In contrast, HDLCAD failed to increase endothelial CAT-1 expression and L-arginine uptake, providing a novel mechanism for altered vascular effects of HDL in patients with CAD.
- © 2010 by American Heart Association, Inc.