Objective: The aim was to determine if isolated rabbit cardiomyocytes could be preconditioned. Methods: Cardiomyocytes isolated from rabbit hearts were subjected to 15 min oxygenated preincubation, with and without substrate, prior to concentration into an ischaemic slurry, with or without glucose present. The effects of an adenosine agonist (CCPA), an adenosine receptor blocker (SPT), and the protein kinase C blocker, calphostin C, on rates of ischaemic contracture and survival of the myocytes were determined after various times of ischaemia, following resuspension of the cells in hypotonic media. Results: A glucose-free preincubation period protected myocytes from subsequent ischaemic injury, with a 40% reduction of cell death at 90-120 min and 1-2 h delay in cell death. CCPA added during preincubation and during the ischaemic period also tended to protect from injury, but the differences were not significant and protection was less than with a glucose-free preincubation. Although preincubation with CCPA did not precondition, SPT added to the preincubation medium only, or to both the preincubation medium and the ischaemic pellet, inhibited the preconditioning effect of a glucose-free preincubation period. Calphostin C, added only into the ischaemic pellet, inhibited the preconditioning effect of glucose-free preincubation. Conclusions: Glucose-free preincubation protects ischaemic isolated myocytes from subsequent ischaemia. The degree of protection is great enough to account for protection seen in intact hearts, following preconditioning protocols. Protection is blocked by SPT and a highly specific protein kinase C inhibitor, calphostin C. Protection from ischaemic injury that seems to mimic ischaemic preconditioning can be induced in isolated cardiomyocytes, and appears dependent on adenosine receptors and activation of protein kinase C.Cardiovascular Research 1994;28:72-77.

Master of Science / Department of Kinesiology / Brett J. Wong / Mechanisms underlying the cutaneous vasodilation response to local skin heating and whole body heating in humans remain unresolved. Although nitric oxide (NO) is known to contribute to these responses, it remains unclear as to the source of NO. Adenosine receptors induce vasodilation in many human tissues and may work, in part, through NO. As these receptors are also known to be located in the cutaneous vasculature, the studies contained in this thesis were designed to investigate a potential contribution of adenosine receptor activation to the rise in skin blood flow elicited by local skin and whole body heating. The study presented in chapter IV was designed to determine a potential role for adenosine receptors in contributing to cutaneous thermal hyperemia. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments designed to elucidate the contribution of A[subscript]1/A[subscript]2 adenosine receptors during local skin heating. Each site was locally heated from a baseline temperature of 33°C to 42°C at a rate of 1°C/10 s and skin blood flow was monitored via laser-Doppler flowmetry (LDF). The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation directly contributes to cutaneous thermal hyperemia. These data further suggest a portion of the NO response may be explained by A[subscript]1/A[subscript]2 adenosine receptor activation; however, a substantial portion of the NO response is independent of the adenosine receptor contribution. The study presented in chapter V was designed to determine a potential role for A[subscript]1/A[subscript]2 adenosine receptors in contributing to cutaneous active vasodilation. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments, as above, and skin blood flow was monitored via LDF. Whole body heat stress, sufficient to raise oral temperature at least 0.8°C above baseline, was induced via water-perfused suits. The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation does not directly contribute to cutaneous active vasodilation; however, a role for A[subscript]1/A[subscript]2 adenosine receptor activation is unmasked when NO synthase is inhibited. The data from this study further suggest that A[subscript]1/A[subscript]2 adenosine receptor activation may be responsible for a portion of the known NO component of cutaneous active vasodilation.

The critical prompt innate immune response is highly built upon the influx of neutrophils from the blood stream to the site of infection. In the battlefield, neutrophils sense pathogen-associated molecular patterns (PAMPs) through their pattern-recognition receptors (PRRs) to launch a number of responses with the goal to defeat the invading pathogen. Neutrophils’ wide spectrum of responses ranges from reactive oxygen species production (ROS), phagocytosis, cytokine and chemokine secretion, and neutrophil extracellular trap (NET) formation. The NET scaffold is composed of nuclear chromatin which is armed with antimicrobial proteins. DNA traps are able to ensnare and kill microbes in the extracellular space and NET release concurs with cell death of the neutrophil. An increasing body of literature describes that NETs impose deleterious effects on the host itself in addition to their antimicrobial activity. These hazardous effects mainly stem from pro-inflammatory and tissue-destructive activity of NETs. These two diverse outcomes of NETs result in a series of effects on both host and pathogen. Therefore, it seems rational that NET formation is tightly regulated and not happening spontaneously. The opportunistic fungal pathogen Candida albicans captured and killed by NETs. This fungus has the remarkable ability to grow as budding yeast or as filamentous hyphae, and reversibly alternate between these morphotypes. Hyphae are the tissue-destructive, invasive and pro-inflammatory form of C. albicans, whereas yeast is the proliferative, non-invasive form. Hence, it is important to find out how neutrophils discriminate between distinct growth forms of C. albicans and how NET release is regulated in this regard. To assess neutrophils responses towards each growth form of C. albicans, the mere ratio of each fungal morphotypes is an insufficient measure to describe comparable amounts used in infection experiments; we therefore used dry mass of fungal cells to serve as a common denominator for amounts of fungal cells with different morphotypes. As assessment of dry mass is laborious, we developed a quick correlative method, which quantified fungal metabolic activity corresponding to the actual dry mass. We applied this method in consecutive studies investigating the neutrophil responses specific to different morphotypes of C. albicans. Positive and negative regulators of NET formation were investigated for this thesis in a mechanistic fashion. To identify how NET release is negatively regulated during C. albicans infection we focused on anti-inflammatory receptors on neutrophils. We observed that adenosine signals via adenosine receptor reduces the amount of NETs exclusively in response to C. albicans hyphae, the invasive, pro-inflammatory form. We identified adenosine receptor A3 as the responsible receptor suggesting that targeting of adenosine A3 would be a promising approach to control invasive fungal infection, since particularly during immune reconstitution invasive mycoses are frequently accompanied by hyperinflammation which additionally worsens the patient’s state. As unbalanced inflammation is harmful to the host, a situation reflected in autoimmune diseases, such as systemic lupus erythematosus, we aimed to find molecules, which are able to inhibit NET formation. Thus, we introduced the non-toxic agent tempol’’. During ROS-depended stimulation of NET formation via C. albicans and phorbol esters, the stable redox-cycling nitroxide tempol efficiently blocked NET induction. We therefore proposed tempol as a potential treatment during inflammatory disorders where NET formation is out of balance. In quest for positive regulators of NET formation we found the major addictive component of tobacco and electronic cigarettes, nicotine, as compelling direct inducer of NET release. Interestingly, nicotine is associated with exacerbated inflammatory diseases exerting its pro-inflammatory activity via acetylcholine receptor by targeting protein kinase B (known as Akt) activation with no effect on NADPH oxidase complex in a ROS independent fashion. In consideration of neutrophils role in smoking-related diseases we propose targeting Akt could lower the undesirable effect of NET.  In conclusion, this thesis identified new modulators of NET formation in response to fungal infection and more broadly to other NET-inducing stimuli, which might have implications in forthcoming therapies.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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by Ai-Dong Qi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 219-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

by Paul Li Wai Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Includes bibliographical references. / Acknowledgements --- p.i / Summary --- p.ii / Index to Figures --- p.V / Index to Tables --- p.vii / Abbreviations --- p.viii / CONTENTS / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter Chapter 2 --- LITERATURE REVIEW : SALT AND HYPERTENSION / Chapter 2.1. --- Summary of evidence linking salt and hypertension --- p.4 / Chapter 2.1.1. --- Epidemiological studies --- p.4 / Chapter 2.1.2. --- Dietary intervention studies --- p.7 / Chapter 2.1.3. --- Experimental studies --- p.9 / Chapter 2.2. --- Cellular sodium transport --- p.10 / Chapter 2.2.1. --- The Sodium Pump --- p.10 / Chapter 2.2.2. --- Defects in sodium transport in hypertension --- p.14 / Chapter 2.3. --- Hypothesis linking salt to the pathogenesis of hypertension --- p.15 / Chapter 2.4. --- Evidence for the presence of natriuretic Hormone --- p.18 / Chapter 2.4.1. --- Indirect evidence --- p.18 / Chapter 2.4.2. --- Direct evidence --- p.18 / Chapter 2.4.3. --- The source and properties of natriuretic hormone --- p.20 / Chapter 2.4.4. --- Other natriuretic factors --- p.21 / Chapter Chapter 3 --- REGULATION OF THE SODIUM PUMP / Chapter 3.1. --- General introduction --- p.24 / Chapter 3.2. --- Regulation of the sodium pump by intracellular sodium --- p.24 / Chapter 3.3. --- "Effects of ethanol on Na+,K+-ATPase activity" --- p.26 / Chapter 3.4. --- "Effects of potassium depletion on Na+,K+-ATPase activity" --- p.27 / Chapter 3.4.1. --- In vivo studies of sodium pump regulation by potassium --- p.27 / Chapter 3.4.2. --- In vitro studies of sodium pump regulation by potassium --- p.29 / Chapter 3.5. --- Effects of cardiac glycosides on the sodium pump --- p.30 / Chapter 3.5.1. --- In vivo studies of sodium pump regulation by cardiac glycosides --- p.31 / Chapter 3.5.2. --- In vitro studies of sodium pump regulation by cardiac glycosides --- p.33 / Chapter 3.6. --- Effects of dietary salt on the sodium pump --- p.35 / Chapter 3.6.1. --- Acute effects of salt-loading --- p.35 / Chapter 3.6.2. --- Chronic effects of salt-loading --- p.36 / Chapter Chapter 4 --- AIMS OF THE STUDY --- p.39 / Chapter Chapter 5 --- MEASUREMENT OF THE SODIUM PUMP ACTIVITY / Chapter 5.1. --- General introduction --- p.41 / Chapter 5.2. --- The measurement of sodium pump activity --- p.42 / Chapter 5.2.1. --- The sodium pump transport activity --- p.42 / Chapter 5.2.2. --- Quantitation of the number of sodium pump sites --- p.45 / Chapter 5.2.3. --- The measurement of enzyme activity --- p.47 / Chapter (a) --- Introduction --- p.47 / Chapter (b) --- Preparation of tissues and detergent treatment --- p.48 / Chapter (c) --- Measurement of ATPase activity by measuring the rate of release of inorganic phosphate --- p.49 / Chapter (d) --- The coupled-enzyme assay --- p.53 / Chapter (e) --- The K+-stimulated 3-0-MFPase assay --- p.54 / Chapter Chapter 6 --- METHODS - ESTABLISHMENT AND EVALUATION / Chapter 6.1. --- Chemicals --- p.57 / Chapter 6.2. --- "Measurement of Na+,K+-ATPase activity by the rate of release of inorganic phosphate" --- p.58 / Chapter 6.3. --- "Automated coupled-enzyme assay of Na+,K+-ATPase activity" --- p.62 / Chapter 6.4. --- "The measurement of Na+,K+-ATPase activity by the potassium-stimulated 3-0-MFPase assay" --- p.67 / Chapter 6.5. --- Determination of protein concentration --- p.70 / Chapter 6.6. --- Statistical analysis --- p.73 / Chapter 6.7. --- Results --- p.73 / Chapter 6.7.1. --- Evaluation of the inorganic phosphate release method --- p.73 / Chapter 6.7.2. --- Evaluation of the coupled-enzyme method --- p.78 / Chapter 6.7.3. --- Evaluation of the K+-stimulated 3-0-MFPase method --- p.89 / Chapter 6.7.4. --- Evaluation of the protein determination method --- p.94 / Chapter 6.8. --- Discussion --- p.96 / Chapter Chapter 7 --- "THE EFFECTS OF DIGOXIN TREATMENT ON Na+,K+-ATPase ACTIVITY OF DIFFERENT TISSUES" / Chapter 7.1. --- Introduction --- p.101 / Chapter 7.2. --- Materials and Methods --- p.103 / Chapter 7.2.1. --- Animals and diets --- p.103 / Chapter 7.2.2. --- Drugs --- p.103 / Chapter 7.2.3. --- Pharmacokinetics of digoxin --- p.103 / Chapter 7.2.4. --- The digoxin regimes --- p.104 / Chapter 7.2.5. --- Preparation and deoxycholate treatment of tissue homogenates --- p.105 / Chapter 7.2.6. --- "Measurement of Na+,K+-ATPase activity" --- p.107 / Chapter 7.2.7. --- Digoxin radioimmunoassay --- p.107 / Chapter 7.2.8. --- Measurement of plasma electrolytes --- p.109 / Chapter 7.3. --- Results --- p.110 / Chapter 7.3.1. --- The pharmacokinetics of digoxin in the rat --- p.110 / Chapter 7.3.2. --- Plasma digoxin levels --- p.110 / Chapter 7.3.3. --- Effects of digoxin treatment on body weight --- p.113 / Chapter 7.3.4. --- Effects of digoxin treatment on plasma electrolytes --- p.117 / Chapter 7.3.5. --- "Effects of digoxin treatment on tissue Na+,K+-ATPase activity" --- p.119 / Chapter 7.4. --- Discussion --- p.129 / Chapter Chapter 8 --- THE SALT-LOADING EXPERIMENT / Chapter 8.1. --- Introduction --- p.140 / Chapter 8.2. --- Materials and Methods --- p.142 / Chapter 8.2.1. --- Animals --- p.142 / Chapter 8.2.2. --- The salt-loading protocol --- p.142 / Chapter 8.2.3. --- Preparation of crude tissue homogenates --- p.143 / Chapter 8.2.4. --- "Measurement of Na+,K+-ATPase activity" --- p.144 / Chapter 8.2.5. --- Analysis of urinary electrolytes --- p.144 / Chapter 8.2.6. --- Measurements of body weight and wet weight of kidney --- p.145 / Chapter 8.3. --- Results --- p.145 / Chapter 8.3.1. --- Effects of salt loading on the body weight --- p.145 / Chapter 8.3.2. --- Effects of salt loading on 24-hour urinary sodium excretion --- p.148 / Chapter 8.3.3. --- Effects of salt loading on the wet weight of kidney --- p.152 / Chapter 8.3.4. --- "Effects of salt loading on tissue Na+,K+-ATPase activity" --- p.152 / Chapter 8.4. --- Discussion --- p.163 / Chapter Chapter 9 --- CONCLUSIONS AND FUTURE WORK --- p.175 / REFERENCES --- p.183

"Oxidative stress is a type of cellular stress that can damage and kill cells. While it is naturally occurring, many non-natural substances found in our environment can also induce the formation of reactive oxygen species (ROS), which then cause oxidative stress within the cell. Oxidative stress has been shown to be involved in the death of neurons in a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The primary causes for these diseases are still unknown; however, we do know oxidative stress plays a primary role in their development. In conditions where oxidative stress is present, adenosine receptor expression has been upregulated and has played a cytoprotective role, but the specific mechanism of action is unknown. In this thesis, oxidative stress was studied in a model eukaryote, Saccharomyces cerevisiae, and the effects of the expression of the human A1 and A2A receptors upon stress response was examined. Oxidative stress was induced by the addition of hydrogen peroxide at concentrations of .5 mM, 1 mM, and 2 mM. The growth of cells expressing either A1-GFP R or A2A-GFP R at the varying hydrogen peroxide concentrations were compared to the parental cells. Confocal microscopy was performed to determine the receptor expression levels, and to confirm the expression of the receptors via their GFP tag. Immunoblots were also performed to assess the receptor expression level at the differing hydrogen peroxide concentrations. A ROS assay was also performed to show the presence of ROS and oxidative stress in the cells. No significant increase in receptor level expression or localization for either A1 R or A2A R at the varying hydrogen peroxide concentrations was found. The data did show trends indicating that A2A receptors may help process the oxidative stress better than A1 receptors and that A2A receptor containing cells had a shorter doubling time. However, more research on this subject should be performed in the future. However, the concentration of hydrogen peroxide should be greatly increased for further experiments in S. cerevisiae in order to better differentiate the trends observed." / 1 / Bryan Goldman