While previous research into the pathogenesis of ischaemic and reperfusion injuries has focussed on the cardiac myocyte, there is increasing evidence for a crucial role for coronary vascular injury in the genesis of the post-ischaemic phenotype [1-3]. Post-ischaemic vascular injury may be manifest as a transient or sustained loss of competent vessels, impairment of vascular regulatory mechanisms, and ultimately as the 'no-reflow' phenomenon (inability to sufficiently reperfuse previously ischaemic tissue despite the removal of the initial obstruction or occlusion). It is now appreciated that the earliest distinguishing feature of various forms of vascular injury (including atherosclerosis and infarction) is 'endothelial dysfunction', which is the marked reduction in endothelial-dependent relaxation due to reduced release or action of endothelial nitric oxide (NO). Importantly, vascular injury may worsen myocardial damage in vivo [4,5], significantly limiting tissue salvage and recovery. The pathogenesis of post-ischaemic vascular injury and endothelial dysfunction is incompletely understood, but has generally been considered to reflect a cardiovascular inflammatory response, neutrophils playing a key role. However, while blood-borne cells and inflammatory elements are undoubtedly involved in the 'progression' of vascular injury [6,7], accumulating evidence indicates that they are not the primary 'instigators' [8]. It should be noted that a wealth of controversial findings exists in the vascular injury literature and mechanisms involved remain unclear. Indeed, multiple mechanisms are likely to contribute to post-ischaemic vascular injury. Adenosine receptors are unique in playing a role in physical regulation of coronary function, and also in attenuating injury during and following ischaemia. While the adenosine receptor system has been extensively investigated in terms of effects on myocardial injury [9,10], little is known regarding potential effects of this receptor system on post-ischaemic coronary vascular injury. This thesis initially attempts to further our understanding of the role of adenosine in normal coronary vascular function, subsequent chapters assess the effect of ischaemia-reperfusion on vascular function, and adenosine receptor modification of vascular dysfunction in the isolated asanguinous mouse heart. Specifically, in Chapter 3 the receptor subtype and mechanisms involved in adenosine-receptor mediated coronary vasodilation were assessed in Langendorff perfused mouse and rat hearts. The study revealed that A2A adenosine receptors (A2AARs) mediate coronary dilation in the mouse vs. A2B adenosine receptors (A2BARs) in rat. Furthermore, responses in mouse involve a sensitive endothelial-dependent (NO-dependent) response and NO-independent (KATP-mediated) dilation. Interestingly, the ATP-sensitive potassium channel component predominates over NO-dependent dilation at moderate to high agonist levels. However, the high-sensitivity NO-dependent response may play an important role under physiological conditions when adenosine concentrations and the level of A2AAR activation are low. In Chapter 4 the mechanisms regulating coronary tone under basal conditions and during reactive hyperaemic responses were assessed in Langendorff perfused mouse hearts. The data support a primary role for KATP channels and NO in mediating sustained elevations in flow, irrespective of occlusion duration (5-40 s). However, KATP channels are of primary importance in mediating initial flow adjustments after brief (5-10 s) occlusions, while KATP (and NO) independent processes are increasingly important with longer (20-40 s) occlusion. Evidence is also presented for compensatory changes in KATP and/or NO mediated dilation when one pathway is blocked, and for a modest role for A2AARs in reactive hyperaemia. In Chapter 5 the impact of ischaemia-reperfusion on coronary function was examined, and the role of A1 adenosine receptor (A1AR) activation by endogenous adenosine in modifying post-ischaemic vascular function was assessed in isolated buffer perfused mouse hearts. The results demonstrate that ischaemia does modify vascular control and signficantly impairs coronary endothelial dilation in a model devoid of blood cells. Additionally, the data indicate that post-ischaemic reflow is significantly determined by A2AAR activation by endogenous adenosine, and that A1AR activation by endogenous adenosine protects against this model of vascular injury. Following from Chapter 5, the potential of A1, A2A and A3AR activation by exogenous and endogenous agonists to modulate post-ischaemic vascular dysfunction was examined in Chapter 6. Furthermore, potential mechanisms involved injury and protection were assessed by comparing effects of adenosine receptors to other 'vasoprotective' interventions, including anti-oxidant treatment, Na+/H+ exchange (NHE) inhibition, endothelin (ET) antagonism, and 2,3-butanedione monoxime (BDM). The data acquired confirm that post-ischaemic endothelial dysfunction is reduced by intrinsic A1AR activation, and also that exogenous A3AR activation potently reduces vascular injury. Protection appears unrelated to inhibition of ET or oxidant stress. However, preliminary data suggest A3AR vasoprotection may share signalling with NHE inhibition. Finally, the data reveal that coronary reflow in isolated buffer perfused hearts is not a critical determinant of cardiac injury, suggesting independent injury processes in post-ischaemic myocardium vs. vasculature. Collectively, these studies show that adenosine has a significant role in regulating coronary vascular tone and reactive hyperaemic responses via NO and KATP dependent mechanisms. Ischaemia-reperfusion modifies vascular control and induces significant endothelial dysfunction in the absence of blood, implicating neutrophil independent injury processes. Endogenous adenosine affords intrinsic vasoprotection via A1AR activation, while adenosinergic therapy via exogenous A3AR activation represents a new strategy for directly protecting against post-ischaemic vascular injury.

The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine) APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 μM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 μM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 μM CCPA. The APNEA curve appeared monophasic, the major slope occurring between 1-100 nM; DPCPX (1 μM) shifted the concentration-response curve ≃ 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of ≃ 5 nM; DPCPX shifted the BNECA concentration-protection relationship ≃ 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.

L’adénosine est un nucléoside ubiquitaire qui exerce un contrôle puissant sur les systèmes nerveux,immunitaire et cardiovasculaire par l’intermédiaire de quatre récepteurs membranaires : A1R, A2AR, A2BR etA3R. L’étude des récepteurs de l’adénosine est nécessaire à la compréhension de physio‐pathologieshumaines non encore élucidées. Pour étudier l’expression des A2AR, nous avons, dans une première étude,produit un anticorps monoclonal, appelé Adonis, d’isotype IgM, . Adonis reconnait un épitope linéaire desept acides aminés sur la partie C‐terminale de la seconde boucle extra‐cellulaire de l’A2AR humain. Adonisrévèle, par Western blotting sur lysats cellulaires, une bande de 45 KDa, correspondant à l’A2AR. Adonis secomporte comme un « agonist‐like » en augmentant la production d’AMPc et en inhibant la proliférationcellulaire via la stimulation des A2AR. Dans une deuxième étude, nous avons utilisé Adonis pour montrerque l’expression des A2AR de cellules mononucléées, qui mime celle des tissus cardiaques, permet dedifférencier certains patients souffrant de syncope neurocardiogénique. Nous avons monté dans unetroisième étude, qu’Adonis induit une « down‐régulation » de l’expression des co‐récepteurs CXCR4 etCCR5 des cellules T via la stimulation des A2AR, et qu’à ce titre il pouvait être un outil thérapeutique dans lesinfections par HIV. Dans une quatrième étude, nous avons évalué les effets anti‐nociceptifs d’Adonis qui,administré par voie intra‐cérébro‐ventriculaire, augmente de manière dose‐dépendante les latencesobtenues avec le test du Hot‐plate et du Tail‐flick chez la souris. Ces effets sont renversés par deuxantagonistes des A2AR mais aussi par un antagoniste des récepteurs aux opioïdes. Ceci suggère que leseffets anti‐nociceptifs d’Adonis sont médiés par la libération d’opioïdes endogènes. En marge de sesétudes, nous avons également testé les propriétés biologiques de nouveaux ligands des A1R dans le cadred’une collaboration entre chimistes et biologistes. Ainsi, nous montrons, dans une cinquième étude, queparmi la trentaine de molécules synthétisées, quatre sont des antagonistes et deux autres des agonistesavec un EC50 de l’ordre du micromolaire pour la production d’AMPc. De tels agonistes des A1R pourraientêtre utiles dans le traitement des douleurs neuropathiques, tandis que les antagonistes le seraient dansl’insuffisance cardiaque ou utilisés comme diurétique. Enfin dans une sixième étude, nous avons testé unemolécule originale, puisque bivalente, possédant un pôle d’activité pour les récepteurs aux opioïdes μ et unautre pour les A1R. Cette molécule est un antagoniste pour les deux récepteurs. Elle pourrait avoir desapplications cliniques dans certaines pathologies comme le choc hypovolémique ou le sevrage aux opiacés. / Adenosine interacts on its cell surface receptors, namely A1R, A2AR, A2BR and A3R, to exertphysiological effects on target tissues. Modulation of these adenosine receptors appears to be a currenttopic of research which may bring more comprehensions on human pathophysiology yet to be elucidated.In order to study A2AR expression, we produced, in study 1, a monoclonal antibody anti‐human A2AR, calledAdonis being of IgM, isotype. Adonis recognized a linear epitope of seven amino acids on the C‐terminalpart of the A2AR second extra‐cellular loop. By Western blotting, Adonis reveals a 45 KDa band of A2AR incell lysates. Adonis behaves as an agonist‐like which increases the cAMP production and inhibits cellproliferation through A2AR stimulation. In study 2, we showed that using Adonis, to measure the A2ARexpression of peripheral blood mononuclear cells which mimic those of the cardiac tissue, was able todifferentiate some patients with suspected neurally mediated syncope. We showed, in study 3, that A2ARstimulation by Adonis leads to a down‐regulation of CXCR4 and CCR5 expression on T‐cells, suggesting thatAdonis would be a potential drug to treat HIV infections. In study 4, we showed that intracereboventricularinjection of Adonis increased the Hot‐plate and Tail‐flick test latencies in mice in a dose‐dependent manner.Such increases were prevented by two A2AR antagonists and by an opiate receptor antagonist, suggestingthat the anti‐nociceptive effects of Adonis were mediated, at least in part, by endogenous opioid liberation.The last section focused on biological evaluation of new A1R ligands in collaborative studies betweenchemists and biologists. Indeed we showed, in study 5, that among thirty synthesized molecules, four act asA1R antagonists and two turn out to be A1R agonists with a micromolar EC50 on cAMP production. ThoseA1R agonists would be used in neuropathic pains, whereas other antagonists could be used in cardiacfailure or as diuretic. Finally, in study 6, we tested an original hybrid molecule which was revealed to be abivalent antagonist to μ opiate receptors and A1R. This hybrid compound may have applications in somepathologies such as hypovolemic shock and opiate addiction.

Mammalian neurons exhibit extreme sensitivity to oxygen deprivation and undergo rapid and irreversible degeneration when oxygen supply is curtailed. Though several neuroprotective pathways are activated during oxygen deprivation, their analyses are masked by the complex series of pathological events which are triggered simultaneously. Such events can be analyzed in the anoxia tolerant fresh water turtle, which can inherently survive the conditions of oxygen deprivation and post-anoxic reoxygenation without brain damage. It is likely in such a model that modulation of a particular molecular pathway is adaptive rather than pathological. The major objective behind this study was to analyze the intracellular signaling pathways mediating the protective effects of adenosine, a potential neuromodulator, and its effect on cell survival by influencing the key prosurvival proteins that prevent apoptosis. In vivo and in vitro studies have shown that adenosine acts as a neuroprotective metabolite and its action can be duplicated or abrogated using specific agonist and antagonists. Stimulating the adenosine receptors using selective A1 receptor agonist N6-cyclopentyladenosine (CPA) activated the presumed prosurvival ERK and P13-K/AKT cascade promoting cell survival, and suppression of the receptor using the selective antagonist DPCPX (8- cyclopentyl-1,3-dipropylxanthine) activated the prodeath JNK and P38 pathways. The complex regulation of the MAPK's/AKT signaling cascades was also analyzed using their specific inhibitors. The inhibiton of the ERK and AKT pathway increased cell death, indicating a prosurvival role, whereas inhibiton of the JNK and p38 pathway increased cell survival in this model. In vitro studies have also shown a high Bcl-2/BAX ratio during anoxia and reoxygenation, indicating a strong resistance to cell death via apoptosis. / Silencing of the anti-apoptotic Bcl-2 gene using specific siRNA upregulated levels of prodeath BAX, thus altering the Bcl-2/BAX ratio and elevating cleaved Caspase-3 levels leading to increased cell death. Another promising neuroprotective target which we analyzed was Neuroglobin, which was induced during oxygen crisis and silencing this gene indicated that its plays a major role in modulation of ROS. This study strongly emphasizes the advantages of an alternate animal model in elucidating neuroprotective mechanisms and revealing novel therapeutic targets which could eventually help clinicians to design new stroke therapies based on naturally tolerant organisms. / by Gauri Nayak. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Wong Lai Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 162-173). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Heterogeneity of mast cells --- p.3 / Chapter 1.3. --- Mast cell mediators --- p.5 / Chapter 1.3.1. --- Performed and granule associated mediators --- p.5 / Chapter 1.3.2. --- Newly synthesized mediators --- p.8 / Chapter 1.3.3. --- Cytokines --- p.10 / Chapter 1.4. --- Mast cell activation --- p.10 / Chapter 1.4.1. --- Aggregation of IgE Receptors (FcεRI) --- p.10 / Chapter 1.4.2. --- Activation of Phospholipase C --- p.11 / Chapter 1.4.3. --- Activation of Adenylate cyclase --- p.13 / Chapter 1.5. --- Adenosine --- p.14 / Chapter 1.5.1. --- Adenosine receptors --- p.14 / Chapter 1.5.2. --- Selective agonists and antagonists --- p.17 / Chapter 1.5.3. --- Physiological and pathological roles of adenosine --- p.20 / Chapter 1.6. --- Role of adenosine receptors in mast cell activation --- p.21 / Chapter 1.7. --- Aims of the study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Mast cells secretagogues --- p.31 / Chapter 2.1.2. --- Anti-allergic compounds --- p.31 / Chapter 2.1.3. --- Adenosine receptor agonists and antagonists --- p.31 / Chapter 2.1.4. --- Materials for buffers --- p.32 / Chapter 2.1.5. --- Materials for rat sensitization --- p.32 / Chapter 2.1.6. --- Materials for histamine assay --- p.33 / Chapter 2.1.7. --- Miscellaneous --- p.33 / Chapter 2.2. --- Buffers and stock solutions --- p.34 / Chapter 2.2.1 --- Buffer ingredients --- p.34 / Chapter 2.2.2 --- Stock solutions --- p.34 / Chapter 2.3. --- Source of mast cells --- p.35 / Chapter 2.3.1. --- Animals --- p.35 / Chapter 2.3.2. --- Sensitization of animals --- p.35 / Chapter 2.3.3. --- Isolation of rat peritoneal mast cells --- p.35 / Chapter 2.3.4. --- Mast cells purification --- p.36 / Chapter 2.3.5. --- Cell counting --- p.36 / Chapter 2.4. --- General protocol for histamine release --- p.37 / Chapter 2.4.1. --- Histamine assay --- p.37 / Chapter 2.4.2. --- Antagonist studies --- p.38 / Chapter 2.4.3. --- Determination of histamine contents --- p.38 / Chapter 2.4.4. --- Calculation of histamine levels --- p.39 / Chapter 2.5. --- Statistical analysis --- p.40 / Chapter Chapter 3 --- "Effects of adenosine, adenosine deaminase and adenosine receptor agonists on mast cell activation" --- p.42 / Chapter 3.1. --- Introduction --- p.43 / Chapter 3.2. --- Materials and methods --- p.44 / Chapter 3.3. --- Results --- p.45 / Chapter 3.3.1. --- Effects of adenosine on anti-IgE induced histamine release in HEPES buffer --- p.45 / Chapter 3.3.2. --- Effects of NECA on anti-IgE induced histamine release in HEPES buffer --- p.46 / Chapter 3.3.3. --- Effects of CCPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.4. --- Effects of CPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.5. --- Effects of CGS21680 on anti-IgE induced histamine release in HEPES buffer --- p.48 / Chapter 3.3.6. --- Effects of Cl-MECA on anti-IgE induced histamine release in HEPES buffer --- p.49 / Chapter 3.3.7. --- Effects of adenosine deaminase on anti-IgE induced histamine release from rat peritoneal mast cells --- p.50 / Chapter 3.3.8. --- Effects of NECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.50 / Chapter 3.3.9. --- Effects of Cl-MECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.53 / Chapter 3.3.10. --- Effects of CV1808 on anti-IgE induced histamine release in HEPES buffer --- p.55 / Chapter 3.4. --- Discussion --- p.76 / Chapter 3.5. --- Conclusion --- p.83 / Chapter Chapter 4 --- Effects of adenosine receptor antagonists on mast cell activation --- p.88 / Chapter 4.1. --- Introduction --- p.89 / Chapter 4.2. --- Materials and methods --- p.90 / Chapter 4.3. --- Results --- p.91 / Chapter 4.3.1. --- Effects of A1 receptor antagonist DPCPX on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.2. --- Effects of A2A receptor antagonist ZM241385 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.3. --- Effects of A2B receptor antagonist MRS 1706 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.92 / Chapter 4.3.4. --- Effects of A3 receptor antagonist VUF5574 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.93 / Chapter 4.3.5. --- Further characterization of adenosine mediated potentiation of anti-IgE histamine release using VUF5574 and ZM241385 --- p.93 / Chapter 4.3.6. --- Effects of theophylline on anti-IgE induced percentage potentiation in HEPES buffer --- p.95 / Chapter 4.4. --- Discussion --- p.130 / Chapter 4.5. --- Conclusion --- p.135 / Chapter Chapter 5 --- Further characterization of the effects of adenosine on mast cells --- p.138 / Chapter 5.1. --- Introduction --- p.139 / Chapter 5.2. --- Materials and methods --- p.141 / Chapter 5.3. --- Results --- p.142 / Chapter 5.3.1. --- Effects of anti-IgE induced histamine release in calcium free and HEPES buffers --- p.142 / Chapter 5.3.2. --- Effects of adenosine on anti-IgE induced histamine release in calcium free buffer --- p.143 / Chapter 5.3.3. --- Effects of adenosine deaminase on compound48/80 induced histamine release from rat peritoneal mast cells --- p.143 / Chapter 5.3.4. --- Effects of adenosine on compound 48/80 induced histamine release in HEPES buffer --- p.144 / Chapter 5.3.5. --- Effects of adenosine deaminase on A23187 induced histamine release from rat peritoneal mast cells --- p.144 / Chapter 5.3.6. --- Effects of adenosine on calcium ionophore A23187 induced histamine release in HEPES buffer --- p.145 / Chapter 5.3.7. --- Effects of adenosine receptor antagonists on inosine mediated enhancement of anti-IgE induced histamine release --- p.145 / Chapter 5.4. --- Discussion --- p.157 / Chapter 5.5. --- Conclusion --- p.160 / References --- p.162

Darbo tikslas: Nustatyti adenozino receptorių (AR) genų raišką (informacinės RNR lygyje) plaučių kraujagyslių endotelio ląstelėse ir žiurkės plaučiuose, jų pokyčius esant hipoksijai, įvertinti AR poveikį endotelio ląstelių proliferacijai. Darbo uždaviniai: 1) Ištirti žiurkės plaučių smulkių kraujagyslių endotelio (PSKE) ir žiurkės plaučių arterijos endotelio (PAE) AR genų raišką, veikiant hipoksijai. 2) Nustatyti AR agonistų poveikį PSKE ląstelių proliferacijai. 3) Nustatyti AR genų raiškos pokyčius žiurkės plaučiuose esant hipoksijai. 4) Nustatyti adenozino receptorių A3 (A3R) pasiskirstymą plaučių arterinės hipertenzijos (PAH) paciento plaučių mėginyje, taikant imunohistocheminį tyrimą. Darbo metodai: genų raiška nustatyta taikant kiekybinę tikro laiko polimerazės grandininę reakciją (KTL-PGR) naudojant “Taqman®” pradmenis ir zondus adenozino receptoriams. Ląstelių proliferacija įvertinta tričiu žymėto timidino (3H-timidino) įjungimu į ląstelių DNR. A3R pasiskirstymas PAH paciento plaučiuose įvertinamas, taikant imunohistocheminį tyrimą. Tyrimo rezultatai: PSKE ląstelėse rasta adenozino receptorių A2B ir A3R. Hipoksijos aplinkoje A2BR genų raiška padidėjo 2 kartus po 24 val., 5 kartus po 40 val. A3R genų raiška sumažėjo 2 kartus po 24 val., 6 kartus po 40 valandų. PAE ląstelėse rasta AR: A1, A2AR ir A2BR. Hipoksijos aplinkoje A1R genų raiška padidėjo 2,5 karto po 24 val. ir išliko padidėjusi po 40 val. A2AR genų raiška padidėjo 2 kartus po 24 val. ir išliko padidėjusi... [toliau žr. visą tekstą] / Aims: Determine adenosine receptors (AR) gene expression in rat pulmonary microvascular endothelial cells (RPMVEC), rat pulmonary artery endothelial cells (RPAEC) and rat lungs during hypoxic conditions. Evaluate effects of adenosine receptors agonist to PRMVEC proliferation. Objectives: 1) Identify AR in RPMVEC and RPAEC. 2) Evaluate AR changes in RPMVEC and RPAEC during hypoxic conditions. 3) Determine adenosine receptors expression in rat lungs exposed to chronic hypoxia. 4) Perform immunohistochemical staining of A3R on a lung section from patient with pulmonary arterial hypertension (PAH). Methods: Adenosine receptors gene expression was determined by quantitative real time polymerase chain reaction (qRT-PCR) assay using “TaqMan®” primers. Cell proliferation was determined using a tritium labeled thymidine (3H-thymidine) assay. Immunohistochemistry was performed on paraffin embedded lung tissue sections. Results: RPMVEC express A2BR and A3R. During hypoxic conditions A2BR was upregulated 2-fold after 24 h. and 5-fold after 40h of hypoxic exposure. A3R was downregulated 2- fold after 24h. and 6-fold after 40h. RPAEC express A1R, A2AR and A2BR. During hypoxic conditions A1R expression was increased 2,5-fold after 24h and 40h. A2AR was upregulated 2-fold after 24h and 40h. A2BR expression was increased 2,5-fold after 24h and 40h of hypoxic exposure. The A3R agonist HEMADO treatment for 24h at the concentration of 10-7 M, increased RPMVEC proliferation 1,5-fold. AR... [to full text]

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during 'metabolic' preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U·ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the A1/A3 antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during "metabolic" preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U · ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.