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.

Oxidative stress (OS) plays a critical role in the pathogenesis of atherosclerosis potentially through interaction with nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) pathway. Although considerable literature supports a pro-atherogenic role for iNOS-induced NO, recent evidence suggest an anti-atherogenic property for this enzyme where iNOS-induced NO attenuates atherosclerotic lesions after immune injury, enhancing endothelial integrity, survival, protecting against OS-induced apoptosis and necrosis. We therefore hypothesize that iNOS may have a cardio-protective role in the atherosclerotic vessel and that under conditions of OS, expression and function of this enzyme may be impaired, thus contributing to the deleterious consequences of OS. Experiments have therefore been conducted to establish whether pro-oxidants regulate iNOS expression/function in rat cultured aortic smooth muscle cells (RASMCs). These cells were induced for 24 hours with LPS and IFN-γ to mimic inflammatory conditions. Oxidative stress inducers may modulate iNOS-induced NO production through alteration of the expression and/or function of the inducible L-arginine-NO pathway. We examined the effects of hydrogen peroxide (H2O2), antimycin A and diethyl maleate (DEM) on this pathway in vascular smooth muscle cells. H2O2 had little effect on NO production or L-arginine transport while antimycin A and DEM independently caused a concentration dependent inhibition of both processes. Only DEM induced hemeoxygenase-1 (HO-1) expression, monitored by western blotting as a marker of OS. The effects of statins on NO synthesis and L-arginine transport in the presence and absence of OS were also investigated. The benefits of statins therapy in cardiovascular medicine are ascribed in part to their lipid-lowering effect by inhibiting 3-hydroxy-3-methoxyglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme for cholesterol synthesis. However, statins may possess anti-inflammatory properties and are able to improve endothelial function, stabilize atherosclerotic plaque, and inhibit platelet aggregation, vascular smooth muscle cells proliferation and vessel wall inflammation. These effects may be exerted through novel actions of statins that include interaction with specific signalling pathways in cells which may be associated with the induction of iNOS and/or cationic amino acid transporters (CATs). Thus, we have extended our investigations to include an examination of the effects of statins on both iNOS and CAT function and expression under control conditions and following exposure of cells to OS. Atorvastatin caused a bell shaped response on NO production and iNOS expression and also enhanced L-arginine transport but in a non-concentration dependent manner. Simvastatin only affected NO synthesis without altering transporter activity. Pravastatin was without effect on either system. Further studies demonstrated that that atorvastatin was able to reverse the effects of antimycin A and DEM but only on NO production. These findings confirm that the inducible L-arginine-NO pathway can be downregulated by pro-oxidants. This mechanism may therefore contribute to the deleterious effects observed in disease states associated with OS. Moreover, statins (in particular atorvastatin) appear to be effective in reversing the inhibition of NO production caused by inducers of OS. This, together with the fact that atorvastatin and simvastatin can potentiate iNOS-induced NO production and indeed L-arginine transport (with atorvastatin), highlights a potential novel mechanism through which the cardio-protective actions of these compounds could be mediated.

The production of Nitric Oxide (NO) under physiological conditions has beneficial roles in acting as a key signaling component of many biological processes as well as having an anti-microbial effect. However its effects following excess production by the inducible NO pathway is potentially detrimental in the pathogenesis of chronic inflammation including sepsis and several other inflammatory diseases. Understanding the mechanisms that regulate the expression of the inducible nitric oxide synthase (iNOS) responsible for producing the excessive amounts of NO in disease states is therefore critical. In this regards, experiments were carried out to identify the signaling pathways that may mediate this process, focusing specifically on the JAK/STAT cascade. The reason for selecting the latter is because our research group, amongst others, has carried out extensive work investigating other signaling pathways, including the mitogen activated kinases (MAPK). Moreover, studies have also been carried out in an attempt to identify the critical role of JAK/STAT signaling for iNOS induction. These studies however failed to conclusively demonstrate whether, as with the MAPKs, the JAK/STATs may also play an essential role. Furthermore there is indeed controversy in the literature with researchers unable to agree whether expression of iNOS does require JAK/STAT activation. Thus, the aim of the project described in this thesis was to establish unequivocally whether activation of the JAK/STATs preceeds induction of iNOS. The studies were extended to L-arginine transport as well because the latter is widely reported to be induced in parallel with iNOS and substrate supply to iNOS may be critical for sustained NO production. Changes in transporter activity as well as their expression profiles were assessed. All experiments were carried out in either rat aortic smooth muscle cells (RASMCs) or in the J774 macrophage cell line. These cell types were selected because RASMCs are one of the prime targets for induced NO production in vascular inflammation and the macrophages are involved in host defence, acting in part through NO production. To establish the role of JAK/STATs, pharmacological and molecular approaches were used. Pharmacologically, two inhibitors were used and these were AG490 and JAK inhibitor I. The former is reported to be a selective JAK2 inhibitor and the other blocks all known JAK proteins. The potential of the GTPases to regulate the induction of iNOS was also examined using selective inhibitor known to regulate these proteins. In addition to these drugs, siRNA targeting JAK2 was also exploited and western blotting was extensively used to detect expression of various proteins including iNOS, native and phosphorylated JAK2 and TYK2. Changes in iNOS activity was monitored by determining nitrite production using the Griess assay and L-arginine transport was monitored using tritiated arginine (L-[3H]arginine). RASMCs were treated with a combination of LPS (100 µg/ml) and IFN- (100 U/ml) and the macrophages with LPS (1 µg/ml) to induce iNOS and transporter activity. Consistent with previous reports, the above treatment of both cell types resulted in the expression of iNOS, production of NO and enhanced transport of L-arginine. These effects were not affected by AG490 but blocked by JAK inhibitor I. Furthermore, although both cell types expressed the key JAKs (JAK2 and TYK2), neither of these proteins were phosphorylated under conditions of induced NO production. Moreover, siRNA experiments showed that JAK2 expression could be abolished without any significant change in NO production, confirming that at least JAK2 may not be required for this process. Whether TYK2 is involved still remains to be resolved as the phosphor-protein could not be detected. However the conclusive siRNA knockdown studies could not be carried out due to time and cost constraints. Apart from iNOS and NO production, changes in induced L-arginine transport were also not significantly affected under the experimental conditions described above suggesting that like with iNOS, induction of L-arginine transport is independent of at least JAK2. Interestingly however, STAT-1 was phosphorylated and this was blocked by JAK inhibitor I but not AG490. Thus, STAT-1 activation may be essential but its activation may be independent of the JAKs. One possible alternate upstream activator of STAT-1 may be the GTPases. Indeed these proteins have been indicated to phosphorylate STAT-1 independent of the JAKs. However, in this project, inhibition of the GTPase pathway enhanced NO production and L-arginine transport suggesting that the GTPases downregulate these processes. In conclusion, the studies carried out in this thesis have shown that induction of iNOS, NO production and L-arginine transport in both RASMCs and J774 macrophages are independent of JAK2 but require STAT-1 activation which may be phosphorylated independently of the JAKs. The role of other JAKs such as TYK2 although unlikely, will need to be resolved using a more specific approach such as siRNA.

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.