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

Abstract

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 μmol/L, 10 s, 37 °C) in the absence or presence of 200 μmol/L N-ethylmaleimide (NEM) (system y⁺/CATs inhibitor) or 2 mmol/L l-leucine (systemy⁺L substrate) was measured. Protein abundance for eNOS and serine¹¹⁷⁷ or threonine⁴⁹⁵ 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 serine¹¹⁷⁷ phosphorylation. Thus, system y⁺L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs.

Keywords: Endothelium; Intracellular pH; System y(+); System y(+)L; l-Arginine transport.