Regulation of Amino Acid Arginine Transport by Lipopolysaccharide and Nitric Oxide Synthesis in Intestinal Epithelial IEC-6 Cells
QingHe Meng, The Pennsylvania University College of Medicine, Hershey, PA; Haroon M. Choudry, Anne M. Karinch, ChengMao Lin, Ming Pan, The Pennsylvania State University College of Medicine, Hershey, PA
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, relationship between intestinal arginine transport and nitric oxide synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and nitric oxide synthesis on the arginine transport in cultured rat intestinal epithelial IEC-6 cell.
Near-confluent IEC-6 cells were incubated with LPS (0 – 25 ug/ml) in serum-free DMEM, in the presence and absence of NO donor sodium nitroprusside (SNP 0 - 50 uM) and inducible nitric oxide (iNOS) synthase inhibitor N-w-nitro-L-arginine (NNA 0 - 500 uM) for various periods of time (0 - 48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured using transport assay, Northern Blot and Western Blot, respectfully. Data were analyzed by ANOVA with p< 0.05.
Lipopolysaccharide (LPS) increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hrs, 25 ug/ml) resulted in a 3-fold increase of arginine transport activity (Control: 28 ± 5; LPS: 92 ± 20 pmole/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 pmole/mg/min, p<0.05) without affecting the transport affinity (Km, Control: 76 ± 8; LPS: 84 ± 14 µM, p=NS). The LPS-induced 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 to control). In contrary, the LPS-induced arginine transport activity was further augmented by N-w-nitro-L-arginine (NNA) (Control: 18 ± 3.2; LPS: 59 ± 2.7*; NNA: 26.3 ± 5.8; LPS+NNA: 127 ± 18+ pmole/mg/min. *p<0.01 compared to control and +p<0.01 compared to control or LPS).
Lipopolysaccharide 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 and nitric oxide synthesis.
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