Arginine transport is primarily mediated by the cationic amino acid transporters (CATs) in mammalian cells, but in aves the y+, b0,+ and B0,+ transport systems have also been observed. Arginine is the limiting catabolic substrate required for the production of nitric oxide (NO), a highly reactive compound that acts as a signaling molecule or killing compound. NO is synthesized by inducible nitric oxide synthase (iNOS) by macrophages for pathogen clearance. In mammals, CAT-2B is responsible for ARG import in the macrophage for NO synthesis, but the chicken CAT-2B isoform does not transport ARG. Therefore the objective of these studies was to identify the CAT(s) involved in mediating ARG uptake during a NO response in the chicken macrophage. Experiments were performed to measure: 1) ARG transporter mRNA and NO production from three sources of macrophages (HD11 cell line, n=6; primary 32d Cobb 500, n=8; Hyline W36, n=7) in response to Escherichia coli lipopolysaccharide (LPS); 2) the effect of CAT over-expression on NO production in response to LPS (HD11 cell line; n=8). In response to LPS iNOS mRNA abundance increased (P<0.05) 8.5-fold in the HD11 macrophages, 3.22-fold in broiler macrophages and 2.79-fold in layer macrophages. In all cells, CAT-1 was induced and CAT-2A increased (P<0.05) between 1.28 and 1.68-fold. CAT-2B was not detected at any time point or treatment condition. In the virally transformed chicken macrophage cell line (HD11) CAT-3 mRNA was induced, but in primary cells CAT-3 increased (P<0.05) 1.27-fold in broilers and 1.23-fold in layers. Transiently transfected chicken macrophages produce NO independent of LPS treatment by 6h, mock transfected controls did not respond by 6h. In the presence of LPS, CAT-1 transfected macrophages produced 50.0% more NO than mock transfected cells (P<0.05). CAT-2A and CAT-3 transfected macrophages produced only 17.6% and 72.1% of the total NO produced by controls (P<0.05). These results indicate that CAT-1 and CAT-3 are both sufficient to sustain ARG import for NO production in the chicken macrophage, but that CAT-1 produces a maximal response. These results also show that iNOS, despite its name, is constitutively present and can be activated by induction of CATs to import ARG.
| Identifer | oai:union.ndltd.org:CALPOLY/oai:digitalcommons.calpoly.edu:theses-1517 |
| Date | 01 April 2011 |
| Creators | Moulds, Michael |
| Publisher | DigitalCommons@CalPoly |
| Source Sets | California Polytechnic State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Master's Theses |
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