Regulation of amino acid arginine transport by lipopolysaccharide and nitric oxide in intestinal epithelial IEC-6 cells

摘要

As a precursor for nitric oxide (NO) synthesis and an immune-enhancing nutrient, amino acid L-arginine plays a critical role in maintaining intestine mucosal integrity and immune functions in sepsis. However, the relationship between intestinal arginine transport and NO synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and NO on the arginine transport in cultured rat intestinal epithelial IEC-6 cell. Near-confluent IEC-6 cells were incubated with LPS (0-50 μg/ml) in serum-free Dulbecco’s modified Eagles’s medium, in the presence and absence of the NO donor sodium nitroprusside (SNP, 0–500 μmol/L) and the inducible nitric oxide synthase (iNOS) inhibitor N-ω-nitro-L-arginine (NNA, 0–1000 μmol/L) for various periods of time (0-48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured with transport assay, Northern blot analysis, and Western blot analysis, respectfully. LPS increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hours, 25 μg/ml) resulted in a 3-fold increase of arginine transport activity (control: 28 ±5; LPS: 92 ±20 pmol/mg/ min, P < 0.05), with the System y+ as the predominant arginine transport system, and a 2-fold increase of System y+ CAT1 mRNA and transporter protein levels (P < 0.05). LPS increased the arginine transport System y+ maximal velocity (Vmax, control: 1484 ±180; LPS: 2800 ±230 pmol/mg/min, P<0.05) without affecting the transport affinity (Km, control: 76 ±8; LPS: 84 ±14 μmol/L, p = NS). The LPSinduced arginine transport activity was blocked by sodium nitroprusside (SNP) (control: 25 ±6; LPS: 97 ±26*; SNP: 22 ±0.4+; LPS+SNP: 33 ±10.3+ pmole/mg/min, *P < 0.01 and +p = NS, compared with control). In contrary, the LPS-induced arginine transport activity was further augmented by NNA (control: 18 ±3.2; LPS: 59 ±2.7*; NNA: 26.3 ±5.8; LPS + NNA: 127 ±18+ pmol/mg/min; *P < 0.01 compared with control and +P < 0.01 compared with control or LPS). LPS-stimulates arginine transport activity in IEC-6 cells via a mechanism that involves increase of transport System y+ mRNA levels and transporter protein levels. The LPS-stimulated arginine transport activity is regulated by the availability of nitric oxide.