Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO). A cytochrome P450-like oxygenase, it uses two monooxygenation steps to convert L-arginine (L-arg) first to N -hydroxy-L-arginine (NOHA), a stable intermediate, and then to L-citrulline and NO. Mammalian NOSs are homodimeric enzymes. Each monomer is composed of an oxygenase domain (containing the L-arg binding site, a heme ligated by a cysteine thiolate, and a tetrahydrobiopterin (H4B)) and a reductase domain (binding NADPH, FAD, and FMN). NOS substrates are O2, L-arg, and NADPH. NADPH is the source of electrons required for oxygen activation. H4B is a vital cofactor that aids dimerisation and acts as a reducing/oxidising agent. Controversy still exists as to the final oxygenating species in the NOS mechanism, but the general reaction scheme is known. The ferric heme is reduced to the ferrous state by an electron from the reductase domain. Then oxygen binds to form the oxy-ferrous species. Then H4B donates an electron to form a peroxy-ferric species. It is likely this then forms a compound 1 (Fe(IV)+.=O) species that is the final oxygenating species. This thesis probes the mechanism of NOS to further define the mechanistic intermediates involved. The role of H4B in NO synthesis has been probed in both normal turnover conditions and special case reactions. To elucidate this mechanism further a mutant with a residue capable of stabilising the activated oxygen species was created, G586S, where glycine 586 of nNOS was replaced with a serine. This serine was within hydrogen bonding distance of the oxy-heme. A stabilised intermediate was observed by stopped flow reaction in the presence of H4B, but not aH4B (an inactive pterin analogue). Here single turnover reactions, each following either the reaction of L-arg to NOHA or NOHA to citrulline, were performed on the mutant using an external source of electrons. The reaction products were observed by HPLC. The mutant appears capable of the conversion of NOHA to citrulline, but not L-arg to NOHA. The WT enzyme appears capable of both. The intermediate is observed with either L-arg or NOHA bound, suggesting both reactions proceed via the same active oxygenating species. The inability of the mutant to catalyse the conversion of L-arg to NOHA may be due to protonation of the substrate hindering reaction such that the active oxygenating species decays before reaction can occur. This mutation, in allowing separation of the two monooxygenation steps, deserves further study. H4B binds at the dimer interface of NOS. Here the -systems of the pterins are only 13Å apart. This is within allowed distances for efficient electron transfer. Electron transfer between hemes, via the pterins, would allow a route for the breakdown of a dead end, ferrous-NO, species. Stopped flow monitoring of the decay of the ferrous-heme NO complex with nNOSoxy dimers with varying proportions of the hemes in the ferrous heme-NO complex showed no electron transfer between hemes of the dimer. The rate of decay of the ferrous heme-NO complex in oxygenated buffer is 0.12 s-1 for all conditions tested here. H4B-deficiency leads to several diseases. H4B makes a poor drug due to instability and cost, the search for druggable analogues of it is ongoing. H4B analogues blocked at the 6,7-positions in the dihydropterine-form have been screened here for catalytic activity. Several have shown comparable ability to catalyse NO production in vitro. Structure function analysis of these analogues has revealed the extent extension is tolerated at the C6 and C7 positions of the pterin.

Rat neuronal nitric oxide synthase (nNOS) is a flavo-hemoprotein that catalyses the NADPH and O2-dependent conversion of L-arginine (L-arg) to L-citrulline and nitric oxide (NO) via the intermediate N-hydroxyarginine. nNOS is a homodimer, where the subunits are modular and are comprised of an N-terminal oxygenase domain (nNOSoxy) that binds iron protoporphyrin IX (heme), (6R)-5,6,7,8-tetrahydro-biopterin (H4B) and L-arg, and a C-terminal flavoprotein or reductase domain (nNOSred) that binds NADPH, FAD and FMN. Regulation of NO biosynthesis by nNOS is primarily through control of interdomain electron transfer processes in NOS catalysis. The interdomain electrons transferred from the FMN to the heme domain are essential in the delivery of electrons required for O2 activation (which occurs in the heme domain) and the subsequent NO synthesis by NOS. Both spectroscopic and kinetic approaches have been used in studying the nature and control of interdomain electron transfer, reaction mechanism and structural changes during catalysis in WT and R1400E nNOS in both full length (FL) and nNOSred. Cytochrome c reduction activity of nNOS was used to determine kinetic parameters for NADPH for FL and nNOSred, WT and R1400E nNOS in the presence and absence of calmodulin (CaM). FL nNOS, where both domains (nNOSred and nNOSoxy) were present, was proven to be more stable and more catalytically efficient than nNOSred by itself. Additionally it was observed that R1400E is still promoting electron transfer despite being thought to lower the affinity of the enzyme to the substrate (NADPH); R1400E also showed lower catalytic efficiency and lower dependence on CaM/Ca2+ compared to the WT. The structure of the functional output state has not yet been determined. In the absence of crystallographic structural data for the NOS holoenzyme, it was important to experimentally determine conformational changes and distances between domains in nNOS. A pulsed EPR spectroscopy (PELDOR) approach has been utilised to gain important and unique information about the conformational energy landscape changes in nNOS. In the presence of CaM, PELDOR results for FL WT nNOS shows a complex energy landscape with multiple conformational states, while in the absence of CaM the interflavin distance distribution matches that exhibited by nNOSred CaM- in the presence of NADP+, suggesting that CaM binding affects some major large-scale conformational changes which are involved in internal electron transfer control in nNOS. A high-pressure stopped-flow technique was also used to perturb an equilibrium distribution of conformational states, to observe the effect of the pressure on the internal electron transfer and to study the kinetics of NADPH oxidation, flavin reduction by NADPH and NO formation. It was shown that high pressure is forcing major changes in the conformational energy landscape of the protein, affecting internal electron transfer. NO formation studies under pressure show that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction, but it has no effect on the heme reduction step.

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.

Abstract Mediators of epithelial permeability, angiogenesis and invasion may serve as prognostic indicators and targets for therapies in esophageal cancer (EC). The expressions of claudins, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor-A (VEGF), nitric oxide synthases (iNOS, eNOS, nNOS) and toll-like receptor 9 (TLR9) were evaluated by immunohistochemistry in EC. The results were compared with clinicopathological variables, tumor proliferation and apoptosis. All of the claudins were expressed in most of the cancers. Esophageal adenocarcinomas (EAC) displayed more often increased claudin 3 and 5 expression than esophageal squamous cell carcinomas (ESCC). Loss of claudin 3 expression associated with distant metastases in EC and a tendency in this direction was also observed for claudin 4. Cancers with stronger claudin 4 expression showed increased apoptosis in both EAC and ESCC. HIF-1α was present in most of the ECs and like iNOS more often in ESCC than in EAC. Strong HIF-1α expression tended to associate with positive VEGF immunostaining. In ESCC, both strong HIF-1α expression and VEGF positivity tended to associate with increased microvessel density (MVD). In EAC, tumors showing VEGF positivity associated with increased MVD outside the tumor. Patients with strong HIF-1α expression had more often distant metastases than other patients in EC. There was no VEGF expression in normal esophageal mucosa and T3-4 tumors tended to be more often VEGF positive than T1-2 tumors. The expression of TLR9 was more intensive in dysplasia than in normal epithelium and ESCC and abundant TLR9 expression could serve as a marker of squamous cell high grade dysplasia. Intensive TLR9 expression was associated with higher grade tumors and the presence of nodal and distant metastases in ESCC. EC and its progression may be related to increased angiogenesis regulated by VEGF and HIF-1α. In EC, claudin expression varies along with the histology of the tumor. Claudin expression may be associated with apoptosis or proliferation and contribute to tumor behavior. An association was detected between moderate to strong expression of claudin 3 and a high TLR9 histoscore in ESCC. Altered expression of claudin 3 may result to upregulation of endosomal TLR9. TLR9 may serve as a marker for squamous cell dysplasia and ESCC progression. / Tiivistelmä Solukerrosten läpäisevyyttä, verisuonten uudismuodostusta ja kasvainsolujen liikkuvuutta säätelevät tekijät voivat toimia ruokatorvisyövän ennustetekijöinä ja hoidon kohteina. Tässä tutkimuksessa selvitettiin klaudiinien, hypoksia-indusoituvan tekijän 1α (HIF-1α), verisuonen endoteelin kasvutekijän A (VEGF), kolmen typpioksidisyntaasin (iNOS, eNOS ja nNOS) sekä tollin kaltaisen reseptorin 9 (TLR9) ilmentymistä ja merkitystä ruokatorvisyövässä immunohistokemiallisin menetelmin. Tuloksia arvioitiin suhteessa proliferaatioon ja apoptoosiin. Useimmat syöpäkasvaimet ilmensivät jokaista tutkittua klaudiinia. Ruokatorven rauhassyövässä ilmeni levyepiteelisyöpää useammin klaudiineja 3 ja 5. Klaudiinin 3 vähäinen ilmentyminen oli yhteydessä etäpesäkkeiseen tautiin. Sama suuntaus näkyi klaudiinin 4 kohdalla. Apoptoosia todettiin enemmän kasvaimissa, jotka ilmensivät muita enemmän klaudiini 4:ä. Useimmat syöpäkasvaimet ilmensivät HIF-1α:a. Sekä iNOS että HIF-1α ilmentyivät runsaammin levyepiteeli- kuin rauhassyövässä. Tutkimus viittasi yhteyteen voimakkaan HIF-1α:n ilmentymisen ja VEGF:n ilmentymisen välillä. Voimakas HIF-1α:n ilmentyminen ja VEGF:n ilmentyminen vaikuttivat liittyvän ruokatorven levyepiteelisyövän uudissuonituksen lisääntymiseen. Rauhassyövän lisääntynyt uudissuonitus kasvaimen ulkopuolella saattaa liittyä VEGF:n ilmentymiseen. Potilailla, joiden kasvaimissa HIF-1α:n ilmentyminen oli voimakasta, todettiin etäpesäkkeitä muita useammin. VEGF:a ei todettu normaalissa limakalvossa, ja sen ilmentyminen vaikutti olevan yleisempää syvälle kasvavissa kuin pinnallisissa syövissä. TLR9 ilmentyi voimakkaammin levyepiteelin dysplasiassa kuin normaalissa tai kasvainepiteelissä. Huonosti erilaistuvissa ja levinneissä kasvaimissa TLR9 ilmentyi voimakkaammin kuin muissa kasvaimissa. Ruokatorvisyövän synty ja eteneminen voivat liittyä HIF:n ja VEGF:n säätelemään verisuonten uudismuodostukseen. Klaudiinit saattavat vaikuttaa syövän käyttäytymiseen myös apoptoosin ja proliferaation kautta. Tutkimuksessa todettiin yhteys lisääntyneen klaudiinin 3 ilmentymisen ja voimakkaasti ilmentyvän TLR9:n välillä. Muutos klaudiinin 3 ilmentymisessä saattaa lisätä levyepiteelin läpäisevyyttä johtaen TLR9:n aktivoitumiseen. TLR9 voi vaikuttaa ruokatorven levyepiteelin dysplasian ja syövän syntyyn sekä toimia vaikean dysplasian ja aggressiivisen levyepiteelisyövän merkkiaineena.