09:45 – 10:00 Introduction by the chair
Dr Nicholas Warrick – University of Oxford

10:00 – 10:30 Fluorescence based methods for the measurement of intracellular nitric oxide.
Prof. David Russell, University of East Anglia, UK

10:30 – 11:00 Quantification of Blood and Tissue NO Metabolites by Gas Phase Chemiluminescence: A Guide to NO Man's Land
Dr. Alexandra Milsom, Wales Heart Research Institute, University of Cardiff, UK
The quantification of NO metabolites in biological samples provides valuable information with regards to in vivo NO production, bioavailability and metabolism. NO detection by gas phase chemiluminescence is based on the reaction of ozone with free NO. Several chemical reagents have been developed to liberate NO, the majority of which reduce NO metabolites back to NO in strong acid. This presentation will discuss the various reductive techniques available to researchers, the different methods of sample preparation, analysis techniques, factors affecting sensitivity, and steps to overcome some of the problems associated with measurement in complex biological matrices.

11:00 – 11:25 Morning Tea/Coffee and view posters

11:25 – 11:55 Radiochemical HPLC measurement of nitric oxide synthesis (NOS) activity in vascular tissue
Dr Nicholas Warrick – University of Oxford
Previous assays of Nitric Oxide Synthesis enzymatic activity measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors. We described a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NOS activity within intact vascular tissue. We demonstrate the presence of arginase activity which has the potential to confound this assay and describe the use of N-hydroxy-nor-L-arginine to inhibit arginase and permit the specific detection of NOS activity. We demonstrate that this technique has high specificity and high sensitivity for detection of in situ NOS enzymatic activity in murine and human vascular tissue.

11:55 – 12:25 EPR-based measurement of reactive nitrogen intermediates in human inflammatory diseases
Prof. Paul Winyard, Peninsula Medical School, Universities of Exeter and Plymouth, UK
Nitric oxide production increases at inflammatory sites, due to the induction of iNOS. In rheumatoid arthritis (RA), TNF-alpha appears to be a key “driver” of inflammation, including iNOS upregulation. However, neurogenic inflammation may also be important. The presence of end-products resulting from oxidative/nitrosative stress (e.g. 3-nitrotyrosine) has been well documented in the inflamed joint. However electron paramagnetic resonance (EPR) spectrometry is the only available method for unequivocally identifying specific free radicals in biological systems. We have used EPR spin trapping – as well as other methods – to detect reactive nitrogen intermediates such as S-nitrosothiols in human inflammatory biofluids, including synovial fluid.

12:25 – 12:55 Nitric Oxide Sensors and Their Biomedical Applications
Dr. Xueji Zhang, World Precision Instruments Inc, Sarasota, Florida, USA
This lecture will focus on how to design, fabricate and characterize different NO electrochemical sensors from nanosensor to microchip sensors. Selectivity, sensitivity detection limit, linearity, and stability of the sensors will be discussed. Applications on measurement of NO in vitro and in vivo such as for cell culture, tissue, in live animals and human will be presented. Finally advantages and disadvantages of NO sensors will be addressed in different applications.

12:55 – 13:10 Tour of the BioPark

13:10 - 14:05 Lunch and Poster session

14:05 – 14:35 Understanding nitric oxide synthase inhibition: potency, selectivity and implications for their pharmacology.
Dr. Alan Wallace, AstraZeneca, UK
NO is involved in many physiological processes and in the pathology of many diseases. Intense effort has been made to find selective inhibitors of both the inducible and neuronal NO synthases. The search has been difficult, perhaps because the isoforms are highly conserved. The best known inhibitors are analogues of arginine, but more drug-like compounds have been created. Several of the most selective inhibitors demonstrate time-dependent inhibition. These achieve selectivity by a ‘mechanism-based’ inhibition – binding at the active site, but inactivating the enzyme in a manner that requires NADPH and involves the reductase activity. Several studies have shown that iNOS inhibitors are anti-inflammatory and model data suggests that inhibition of iNOS will be beneficial in several diseases, including osteoarthritis or asthma.

14:35 – 15:05 Endogenously occurring inhibitors of nitric oxide synthase
Dr Caroline Smith, University of Westminster, UK.
The earliest inhibitors of nitric oxide synthase (NOS) were structural analogues of the substrate, arginine. In 1992 endogenously occurring asymmetric dimethylarginine (ADMA) was reported to have an in vivo effect on NO generation1. ADMA inhibits endothelial, neuronal and inducible NOS isoforms.

Increased levels of ADMA have been widely reported in a range of cardiovascular disorders and it is currently regarded as a cardiovascular risk factor. Pathophysiological levels of ADMA elicit changes in gene expression, in vitro and in vivo, which may assist in understanding of cardiovascular pathologies.

Techniques which have been developed to measure ADMA levels will be discussed.

1. Vallance et al 1992 Lancet. 339: 572-5.

15:05 – 15:30 Afternoon Tea/Coffee and last poster viewing

15:30 – 16:00 Evaluating endothelial function in humans: From bench to bedside
Dr. Charalambos Antoniades, Athens, Greece
Evaluation of endothelial function in humans is a challenge faced by all clinicians, since endothelial dysfunction represents the very first step in atherogenesis, and it is largely reversible by a wide range of currently used medications. The first category of techniques evaluating endothelial function in humans, includes ex-vivo evaluation of nitric oxide (NO) bioavailability in human vessels (arteries and veins), obtained during coronary artery bypass grafting operations (CABG). In these vessels, NO bioavailability can be measured directly or indirectly by determining their vasomotor responses to endothelium-specific stimuli, such as acetylcholine, bradykinin, substance P and others. Also, endothelial NO synthase (eNOS) activity can be determined by evaluating the radiolabelled arginine/citrulline conversion, while eNOS ‘coupling’ can also be evaluated by measuring the LNAME-inhibitable superoxide production or even by determining monomer:dimer ratio (by using western blotting). Further to these techniques, endothelial function can also be assessed by a range of invasive techniques in vivo, which include intracoronary or intrabrachial infusions of vascoactive agents inducing endothelium-derived NO synthesis and evaluate the vasomotor responses of these conduits to these stimuli. However, the real challenge is to develop non-invasive techniques to evaluate endothelial function in humans, which could be used as screening tests to detect “healthy” subjects with endothelial dysfunction, and design therapeutic interventions targeting vascular endothelium. Non-invasive techniques, currently available, use shear stress to induce endothelium-dependent vasodilation of peripheral arteries such as the brachial artery, which is visualised by ultrasound or more accurate imaging techniques, such as magnetic resonance imaging. All these techniques have their advantages and disadvantages, and they are used as research tools, based on the demands and the underlying hypothesis of each research project.

16:00 – 16:15 Nitric Oxide Synthase Inhibition and Renal Injury
Prof Sharma Prabhakar, Texas Tech University Health Sciences Center Lubbock, TX, USA
Alterations of renal NO generation have been incriminated in various renal disorders. Acute kidney injury as well as chronic kidney disease especially from diabetes is associated with changes in intrarenal NO generation and/or bio-availability. Manipulation of various NOS isoforms and consequent NO changes have been the focus of investigations aimed at slowing the development and progression of such disorders. Several of the observations made by these studies are not only crucial in understanding the pathophysiology but also provide potentially novel therapeutic targets in various renal disorders.

16:15 – 16:30 Quantification of enzyme independent stores of nitric oxide in human skin
Dr Megan Mowbray, The University of Edinburgh, Scotland
UVR induces up regulation of iNOS in human skin and enzyme dependent NO formation, maximal 8-10hours post exposure. In contrast NO can be stored in human skin in the form of nitrosospecies, providing a non-enzymatic source of NO, these nitrosospecies can be photolysed by UVA to release NO within 20 minutes of exposure.

In 3 separate experiments we analysed biological samples for nitrosospecies content:

Exp 1 - Epidermal suction blisters

Exp 2 - Cutaneous microdialysis (superficial vascular dermis)

Exp 3 - Sweat, saliva and serum

We have shown that important enzyme independent NO stores in human skin can be found in the epidermis and dermis of the skin itself and in sweat on the skin surface.

16:30 – 17:00 Chairman’s summing up & close.

17:15 – 17: 35 *2nd Tour of the BioPark for delegates

*Please note that a tour for exhibitors is scheduled for 2:15- 2:45