Intracellular acidification reduces L-arginine transport via system y(+)L but not via system y(+)/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells

摘要

L-Arginine is taken up via the cationic amino acid transporters (system y(+)/CATs) and system y(+)L in human umbilical vein endothelial cells (HUVECs). L-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y(+)/CATs and system y(+)L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates L-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH4Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, L-arginine transport (0-20 or 0-1000 mu mol/L, 10 s, 37 degrees C) in the absence or presence of 200 mu mol/L N-ethylmaleimide (NEM) (system y(+)/CATs inhibitor) or 2 mmol/L L-leucine (systemy(+)L substrate) was measured. Protein abundance for eNOS and serine(1177) or threonine(495) phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y(+)L but not system y(+)/CATs mediated L-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine(1177) phosphorylation. Thus, system y(+)L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs.