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Y+-LAT-1 and CAT-2B respectively transport GW274150 in control and activated J774 macrophages GW274150 has been identified as a potent inhibitor of the inducible nitric oxide synthase (iNOS) enzyme both in in vivo and in in vitro (Young et al. 2000; Alderton et al., 2005). We have previously demonstrated that this compound may be transported into control J774 macrophages via a broad scope carrier, characteristic of system y+-LAT1 (Baydoun et al., 2006). We now report that activation of these cells with bacterial lipopolysaccharide (LPS) results in a switch in transporters where the inducible cationic amino acid transporter (CAT)-2B becomes the critical carrier system for GW274150. Confluent monolayers of J774 macrophages were pre-treated with LPS (1μg ml-1) for 24 h in Dulbecco's modified Eagle's medium supplemented with 10 % foetal bovine serum. Transport was monitored over 1 min in Hepes-buffered Krebs solution (50μl; 37oC) containing L-[14C]GW274150 (1μCiml-1) and 0.1mM unlabelled compound (Baydoun et al., 2006). Cross-inhibition studies were carried out in the presence of a 10-fold excess (1 mM) of known substrates for different amino acid transports. The Na+-dependency of transport was determined in a modified Na+-free Krebs buffer supplemented with choline chloride and bicarbonate. Kinetic studies were carried out using different substrate concentrations. Activation of J774 macrophages resulted in the expected induction of iNOS expression and in NO synthesis. In addition, the control rate of GW274150 transport (2.6 pmol. μg protein-1 min-1) was significantly enhanced in cells treated with LPS (4.2 pmol. μg protein-1 min-1 ). This upregulation in uptake of GW274150 was further confirmed in kinetic studies, which revealed an enhanced V max of 13.4 pmol. μ protein-1 min-1 from a control value of 8.5 pmol. μg protein-1 min-1. The Kt remained virtually unaltered ( 0.24±0.01 mM in controls vs 0.26±0.02 mM in activated cells). Further characterisation revealed that the uptake process in both control and activated cells was pH insensitive, largely Na+-independent and inhibited by L-arginine and L-lysine but unaffected by 2-methylaminoisobutyric acid, L-alanine, L-valine or ß-2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid. More importantly, transport of L-[14C]GW274150 in control but not in activated cells was markedly attenuated, and in a Na+-dependent manner, by L-leucine, L-methionine, 6-diazo-5-oxo-L-norleucine and L-glutamine. Our previous findings (Baydoun et al., 2006) together with the current data strongly suggest that control and activated J774 macrophages transport GW274150 via different carrier systems. The broad-spectrum amino acid carrier y+-LAT1 may be the critical transporter in controls while CAT-2B may be the logical transporter in activated cells. Further studies looking at changes in expression of y+-LAT1 and CAT-2B in control and activated J774 macrophages are however required and these studies are currently being carried out.
Alderton et al. (2005). Br. J. Pharmacol, 145:301-312. |
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