No / Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N¿-nitro- -arginine methyl ester hydrochloride ( -NAME, 1 mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.

Die Herzinsuffizienz ist trotz zahlreicher Therapiemöglichkeiten immer noch eine der häufigsten chronischen Erkrankung und Todesursachen in westlichen Industrienationen. Eine zentrale Rolle in der Regulation der effizienten Herzkontraktion nimmt die zyklisches Adenosin-3’,5’-monophophat(cAMP)-Signalkaskade ein, wobei Veränderungen in der Kompartimentierung des sekundären Botenstoffes bisher nicht vollständig verstanden sind. Ziel dieser Studie war es deshalb Regulationsmechanismen des lokalen cAMP-Pools der Mikrodomäne der ATP-abhängigen Calciumpumpe 2a des sarkoplasmatischen und endoplasmatischen Retikulums (SERCA2a) in kardialen Mausmyozyten unter den pathologischen Rahmenbedingungen der Herzinsuffizienz zu untersuchen. Hierfür wurde ein post-Myokardinfarkt Mausmodell an einer transgene Mauslinie verwendet, die einen cAMP-abhängigen auf Förster-Resonanz-Energietransfer(FRET)-basierenden Biosensor, lokalisiert in der SERCA2a-Mikrodomäne, in vivo exprimiert. Mit Hilfe von Echtzeit-FRET-Messungen an frisch isolierten, lebenden Kardiomyozyten wurden die Beiträge der am Herzen relevanten Phosphodiesterase(PDE)-Familien zur Begrenzung des lokalen cAMP-Pools in der SERCA2a-Domäne 12 Wochen nach Myokardinfarkt gemessen und mit einer Kontrollgruppe (Sham) verglichen. Hierbei zeigte sich, dass in der Mikrodomäne sowohl unter Ruhebedingungen, als auch nach β-adrenerger Vorstimulation, eine signifikante Aktivitätsminderung der PDE4, verglichen mit der Sham-Gruppe, nachweisbar ist. Da dies mit Veränderungen im lokalen cAMP-Pool der die SERCA2a reguliert einhergeht, bietet diese Studie also eine interessante Grundlage für die weitere Untersuchung der im Krankheitsfall auftretenden Funktionsabweichungen.

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.:Table of contents Abbreviations 1 1. Introduction 2 1.1 The dopaminergic system 2 1.2 Neurite regeneration following mechanical lesions of the CNS 7 1.3 Organotypic brain slice co-cultures 8 1.4 Promising substances and cells to enhance neuroregeneration 10 1.5 The aim of the thesis 14 2. The original research articles 16 2.1 Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures 17 2.2 Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures 35 2.3 Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model 50 3. References 66 Appendices 73 Summary 73 Zusammenfassung 78 Curriculum Vitae 84 Track Record 85 Selbständigkeitserklärung 87 Acknowledgments 88

Background: Pulmonary hypertension (PH) is a rare condition characterized by endothelial dysfunction and vascular remodelling, leading to increased pulmonary vascular resistance (PVR) and right ventricular heart failure. Endothelial dysfunction is associated with an imbalance between vasoconstrictor compounds, such as endothelin and thromboxane A2, and vasodilator compounds, such as prostacyclin and nitric oxide (NO). Asymmetric dimethylarginine (ADMA), a methyl derivate of L-arginine, inhibits synthesis of NO. Vardenafil, a phosphodiesterase type 5 inhibitor (PDE5-inhibitors), causes vasodilation through the NO/cGMP pathway. Aim: This thesis investigates the pharmacological effects and diagnostic utility of vardenafil in PH patients. In addition, to evaluate the change of L-arginine and dimethylarginines before and during PAHspecific therapy in PAH patients compared to patients with left ventricular heart failure (LVHF) and healthy subjects. Methods: The pharmacokinetics and hemodynamic effects of vardenafil were examined during right heart catheterization (RHC) in 16 PH patients and plasma concentrations were measured for up to nine hours after oral administration. In 20 PH patients, acute vasoreactivity test with vardenafil was performed during RHC. Hemodynamic responses were recorded, responders were defined and followed for up to seven years. Additionally, plasma ADMA, symmetric dimethylarginine (SDMA), L-arginine, L-citrulline and L-ornithine levels before and after PAH drug treatment were monitored in 21 PAH patients and compared to values measured in 14 LVHF patients and 27 healthy subjects. Results: Vardenafil concentrations increased rapidly to maximum plasma concentration (tmax 1h) and elimination half-life was 3.4 h. Patients co-medicated with bosentan had reduced vardenafil concentration. Significant acute hemodynamic responses were observed for mean pulmonary artery pressure (mPAP) (p<0.001), pulmonary vascular resistance (PVR) (p<0.001), cardiac output (CO) (p=0.015), cardiac index (CI) (p=0.010), systemic vascular resistance (SVR) (p<0.001) and PVR/SVR (p=0.002) and were related to plasma vardenafil concentrations. PAH patients had significantly higher ADMA and SDMA levels and significantly lower L-arginine levels and L-arginine/ADMA ratio compared with healthy subjects (p<0.001). L-arginine was also lower in PAH patients compared to patients with LVHF (p<0.05). WHO functional class and six minutes walking distance (6MWD) correlated to Larginine and L-arginine/ADMA ratio in PAH at baseline (p<0.05). At follow-up, patients on mono- or combinationtherapy with endothelin receptor antagonists (ERA) had lower ADMA levels than patients without ERA (p<0.05). In contrast, patients on PDE5-inhibitors had higher ADMA levels compared to patients without PDE5-inhibitors (p<0.05). Conclusion: Vardenafil is safe in acute vasoreactivity test in PH patients. Cardiopulmonary hemodynamic response was related to plasma drug concentrations. There was a high inter-individual variability of vardenafil pharmacokinetics and co-medication with bosentan caused a pharmacokinetic drug interaction. Baseline L-arginine and dimethylarginines levels were different in PAH patients compared to LVHF patients and healthy controls. PAH-specific treatment influenced L-arginine and dimethylarginines. Our data suggest that L-arginine might be useful for differentiating PAH from LVHF, and L-arginine/ADMA ratios were related to the severity of PAH and might be useful for follow-up evaluations of PAH patients.

Insulin resistance and chronic hyperglycemia promote vascular damage, increase circulating levels of inflammatory cytokines and lead to increased morbidity and mortality. MicroRNAs (miRs) -103/107 have been shown to negatively regulate insulin sensitivity and glucose homeostasis. Based on complimentary binding profiles, the downstream target gene of miR-103/107 is caveolin-1 (Cav-1). We hypothesized that daily administration of the phosphodiesterase-5 inhibitor tadalafil (TAD) ± the curcumin analogue (HO-3867) will attenuate inflammation, improve metabolic parameters and reduce infarct size after ischemia/reperfusion injury (IRI). Furthermore, we propose that TAD therapy will reduce myocardial expression of miR-103/107 and increase mRNA and protein levels of its target gene, Cav-1. Leptin receptor null mice were randomized to receive daily injections of TAD (1mg/kg), HO-3867 (25mg/Kg), combination therapy, or control for 12weeks with weight and fasting glucose monitored weekly. Upon completion, cardiomyocytes were isolated from each group and were subjected to simulated ischemia and reoxygenation (SI/RO) for cell viability and reactive oxygen species (ROS) measurement. Another set were subjected to IRI in a Langendorff model. Plasma samples were taken to measure plasma concentrations of cytokines. For miR expression, total RNA was isolated from TAD and DMSO treated mice and was subjected to reverse transcription and real time PCR using miR assay probes to determine expression. TAD, HO-3867 and the combination of both attenuated fasting glucose levels, reduced myocardial infarct size after IRI and inflammatory cytokines when compared to control (p<0.05 for each vs. control). Cardiomyocytes isolated from each treatment groups and subjected to SI/RO demonstrated reduced necrosis as shown by trypan blue exclusion assay, ROS generation, and improved mitochondrial membrane potential as compared to DMSO (control). Likewise, both mRNA and protein expression of Cav-1 were reduced in diabetic hearts but were significantly increased in TAD treated diabetic mice, which may be a mechanism to improve insulin signaling through downregulation of miR-103/107 and upregulation of Cav-1. These studies suggest that TAD alone or in combination may be a unique strategy to improve metabolic parameters and precondition diabetic hearts against IRI.

L'AMP cyclique (AMPc) et le GMP cyclique (GMPc) sont des seconds messagers essentiels pour la régulation de la fonction cardiaque. Leurs niveaux sont régulés par l’adénylate cyclase et la guanylate cyclase, respectivement, et par les phosphodiestérases (PDEs). Cependant, une telle régulation est altérée dans l'insuffisance cardiaque (IC). En effet, la diminution de la signalisation de l’AMPc et l’augmentation de celle du GMPc est caractéristique des cœurs défaillants.Parmi la superfamille des PDEs, la PDE2 a la particularité d'être stimulée par le GMPc, conduisant ainsi à une augmentation remarquable de l'hydrolyse de l'AMPc. Ceci semble induire une interaction entre les voies de signalisation de l’AMPc et du GMPc. Cependant, le rôle de la PDE2 dans le cœur défaillant est très peu connu.Dans ce contexte, nous avons examiné si la PDE2 cardiaque est modifiée dans l’IC chez l’Homme et chez les modèles animaux d’IC, et déterminé le rôle de la PDE2 dans la signalisation β-adrénergique dans les cardiomyocytes. Grâce à l’utilisation de Western blot, de technique radioenzymatique, d’imagerie basée sur le FRET, de la planimétrie, de la microscopie à épifluorescence et des mesures du courant calcique de type L, réalisés sur les tissus myocardiques humains et/ou dans des cardiomyocytes isolés de cœurs des modèles animaux d’IC, respectivement, nous avons montré que l’expression et l’activité de la PDE2 sont augmentées dans les cœurs défaillants. Cette augmentation réduit l’effet d’une stimulation β-adrénergique aiguë, contribuant à la désensibilisation β-adrénergique observée dans l’IC. En accord avec ces résultats, la surexpression de la PDE2 dans des cardiomyocytes sains, réduit l’augmentation des taux d'AMPc et l’amplitude du courant ICa,L et abolit l'effet inotrope positif suite à une stimulation β-adrénergique aiguë, sans affecter la contractilité basale. Plus important, les cardiomyocytes surexprimant la PDE2, montrent une protection contre les réponses hypertrophiques induites par la noradrénaline et contre les arythmies induites par l'isoprotérénol.En conclusion, ce travail met en évidence l'altération de la PDE2 dans l’IC et nous laisse suggérer que l’augmentation de la PDE2 dans l’IC peut constituer un mécanisme de défense important dans des conditions de stress cardiaque, notamment en antagonisant la suractivation de la voie β-adrénergique. Ainsi, l'activation de PDE2 myocardique peut représenter une nouvelle stratégie thérapeutique anti-adrénergique intracellulaire dans l’IC. / Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers for the regulation of cardiac function. Their levels are regulated by adenylyl and guanylyl cyclases, respectively, and by cyclic nucleotides phosphodiesterases (PDEs). However, such regulation is altered in heart failure (HF). Indeed diminished cAMP- and augmented cGMP-signaling is characteristic of failing hearts.Among the PDE superfamily, PDE2 has the unique property to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis. This appears to mediate a negative cross-talk between cAMP- and cGMP signaling pathways. However, the role of PDE2 in the failing heart is only poorly understood.In this context, we investigated whether myocardial PDE2 is altered in human and experimental HF and determined PDE2 mediated effects on β-adrenoceptor (β-AR) signaling in cardiomyocytes. Using immunoblotting, radioenzymatic- and FRET-based assays, video-edge-detection, epifluorescent microscopy and L-type Ca2+ current measurements, performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively, we showed that PDE2 is markedly upregulated in failing hearts. This reduces the effect of an acute β-adrenergic stimulation, and contributes to the β-adrenergic desensitization which is a characteristic feature in HF. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and ICa,L amplitude and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses and from isoproterenol-induced arrhythmias.In conclusion, this work highlights the alteration of PDE2 in HF and lets us assume that PDE2 upregulation in HF may constitute an important defence mechanism during cardiac stress, e.g. by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular anti-adrenergic therapeutic strategy in HF.

Kam Man Fai Afia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [195]-224). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The Mast Cell --- p.2 / Chapter 1.1.1 --- Historical Perspective --- p.2 / Chapter 1.1.2 --- Mast Cell Origin and Development --- p.3 / Chapter 1.1.3 --- Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.1 --- Rodent Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.2 --- Human Mast Cell Heterogeneity --- p.7 / Chapter 1.1.4 --- Mast Cell Mediators --- p.10 / Chapter 1.1.4.1 --- Preformed Mediators --- p.11 / Chapter 1.1.4.2 --- Newly Synthesized Lipid Mediators --- p.14 / Chapter 1.1.4.3 --- Cytokines --- p.16 / Chapter 1.1.5 --- Mast Cell Activation --- p.17 / Chapter 1.1.5.1 --- Immunological Activation --- p.19 / Chapter 1.1.5.1.1 --- FcεIR Activation and Protein Tyrosine Phosphorylation --- p.19 / Chapter 1.1.5.1.2 --- Activation of Phospholipases --- p.20 / Chapter 1.1.5.1.3 --- The Role of Calcium --- p.22 / Chapter 1.1.5.1.3.1 --- Intracellular Calcium Mobilization --- p.23 / Chapter 1.1.5.1.3.2 --- Calcium Influx --- p.24 / Chapter 1.1.5.1.3.3 --- Mechanisms of Action of Calcium in Mast Cells --- p.28 / Chapter 1.1.5.1.4 --- The Role of G-proteins --- p.30 / Chapter 1.1.5.1.5. --- The Role of Cylic AMP --- p.33 / Chapter 1.1.5.1.2.1 --- Mechanisms of Action of Cyclic AMP in Mast Cells --- p.36 / Chapter 1.1.5.1.2.2 --- Implications for the Inhibitory Role of Cyclic AMP in Mast Cell Activation --- p.37 / Chapter 1.2 --- The Cyclic Nucleotide Phosphodiesterases --- p.39 / Chapter 1.2.1 --- Introduction --- p.39 / Chapter 1.2.2 --- Classification and Structure --- p.41 / Chapter 1.2.3 --- Distribution and Physiological Functions of the Different PDE Families --- p.45 / Chapter 1.2.4 --- Phosphodiesterase Inhibitors --- p.49 / Chapter 1.2.4.1 --- Non-selective PDE Inhibitors --- p.50 / Chapter 1.2.4.2 --- Selective PDE Inhibitors --- p.52 / Chapter 1.2.4.2.1 --- PDE1 and PDE2 Inhibitors --- p.52 / Chapter 1.2.4.2.2 --- PDE3 Inhibitors --- p.53 / Chapter 1.2.4.2.3 --- PDE4 Inhibitors --- p.54 / Chapter 1.2.4.2.4.1 --- PDE5 Inhibitors --- p.56 / Chapter 2. --- Materials and Methods --- p.59 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Drugs --- p.60 / Chapter 2.1.1.1 --- Phosphodiesterase Inhibitors --- p.60 / Chapter 2.1.1.2 --- Mast Cell Secretagogues --- p.61 / Chapter 2.1.2 --- Materials for Rat Peritoneal Mast Cell Experiments --- p.61 / Chapter 2.1.2.1 --- Materials for Rat Sensitization --- p.61 / Chapter 2.1.2.2 --- Materials for Buffers --- p.62 / Chapter 2.1.2.3 --- Materials for Histamine Assay --- p.62 / Chapter 2.1.2.4 --- Miscellaneous --- p.63 / Chapter 2.1.3 --- Materials for RBL-2H3 Cell Line Experiments --- p.63 / Chapter 2.1.3.1 --- Materials for Cell Culture --- p.63 / Chapter 2.1.3.2 --- Materials for Cell Sensitization and Enzyme Release --- p.64 / Chapter 2.1.3.3 --- Materials for β-Hexosaminidase Assay --- p.64 / Chapter 2.1.3.4 --- Miscellaneous --- p.64 / Chapter 2.2 --- Rat Peritoneal Mast Cell Experiments --- p.65 / Chapter 2.2.1 --- Preparation of Buffers --- p.65 / Chapter 2.2.2 --- Preparation of Stock Solutions --- p.66 / Chapter 2.2.2.1 --- Mast Cell Secretagogue Stock Solutions --- p.66 / Chapter 2.2.2.2 --- Phosphodiesterase Inhibitor Stock Solutions --- p.66 / Chapter 2.2.3 --- Animals and Cell Isolation --- p.71 / Chapter 2.2.3.1 --- Animals --- p.71 / Chapter 2.2.3.2 --- Sensitization of Animals --- p.71 / Chapter 2.2.3.3 --- Cell Isolation --- p.71 / Chapter 2.2.3.4 --- Cell Purification --- p.72 / Chapter 2.2.3.5 --- Determination of Cell Number and Viability --- p.73 / Chapter 2.2.4 --- General Protocol for Histamine Release and Histamine Measurement --- p.75 / Chapter 2.2.4.1 --- Histamine Release --- p.75 / Chapter 2.2.4.2 --- Spectrofluorometric Determination of Histamine Content --- p.76 / Chapter 2.2.4.2.1 --- Manual Histamine Assay --- p.76 / Chapter 2.2.4.2.2 --- Automated Histamine Assay --- p.78 / Chapter 2.2.4.3 --- Calculation of Histamine Levels --- p.78 / Chapter 2.2.4.4 --- Presentation and Statistics --- p.79 / Chapter 2.3 --- RBL-2H3 Cell Line Experiments --- p.80 / Chapter 2.3.1 --- Preparation of Stock Solutions --- p.80 / Chapter 2.3.2 --- Preparation of Materials for Enzyme Release and Assay --- p.81 / Chapter 2.3.2.1 --- Cell Culture --- p.81 / Chapter 2.3.2.2 --- Preparation of Cells for β-Hexosaminidase Release Experiments --- p.82 / Chapter 2.3.2.3 --- β-Hexosaminidase Release --- p.82 / Chapter 2.3.2.4 --- β-Hexosaminidase Assay --- p.83 / Chapter 3. --- Effects of Phosphodiesterase Inhibitors on Mediator Release from Rat Mast Cells --- p.84 / Chapter 3.1 --- Introduction --- p.85 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Rat Peritoneal Mast Cells --- p.87 / Chapter 3.2.1.1 --- Experiments Employing Immunological Stimulus in RPMCs --- p.87 / Chapter 3.2.1.2 --- Experiments Employing Non-Immunological Stimuli in RPMCs --- p.88 / Chapter 3.2.2 --- Rat Basophilic Leukemia Cells --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1 --- Immunologically Activated Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1.1 --- Effects of Non-Selective PDE Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.89 / Chapter 3.3.1.1.2 --- Effects of Selective PDE1 and PDE2 Inhibitors on Anti-IgE- Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.3 --- Effects of Selective PDE3 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.4 --- Effects of Selective PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.1.5 --- Effects of Selective PDE5 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.2 --- Non-Immunologically Activated Rat Peritoneal Mast Cells --- p.92 / Chapter 3.3.1.2.1 --- Effects of Selective PDE Inhibitors on Compound 48/80- Mediated Histamine Release from RPMCs --- p.92 / Chapter 3.3.1.2.2 --- Effects of Selective PDE Inhibitors on Histamine Release from RPMCs Stimulated by Calcium Ionophores --- p.93 / Chapter 3.3.2 --- Rat Basophilic Leukemia Cells --- p.93 / Chapter 3.3.2.1 --- Effects of Non-Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.93 / Chapter 3.3.2.2 --- Effects of Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.94 / Chapter 3.4 --- Discussion --- p.95 / Chapter 3.4.1 --- Rat Peritoneal Mast Cells --- p.95 / Chapter 3.4.1.1 --- Immunologically Activated RPMCs --- p.95 / Chapter 3.4.1.2 --- Non-Immunologically Activated RPMCs --- p.99 / Chapter 3.4.2 --- Rat Basophilic Leukemia Cells --- p.103 / Chapter 4. --- Combined Effects of Selective Phosphodiesterase Inhibitors on Immunologically Induced Histamine from Rat Mast Cells --- p.143 / Chapter 4.1 --- Introduction --- p.144 / Chapter 4.2 --- Materials and Methods --- p.144 / Chapter 4.2.1 --- Simultaneous Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.2.2 --- Sequential Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.3 --- Results --- p.146 / Chapter 4.3.1 --- Effects of the Selective Inhibitors for PDE3 and PDE4 Alone: Calculation of the Expected Inhibition Curve --- p.146 / Chapter 4.3.2 --- Effects of the Simultaneous Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.148 / Chapter 4.3.2.1 --- Rolipram and Siguazodan --- p.148 / Chapter 4.3.2.2 --- Ro 20-1724 and Siguazodan --- p.149 / Chapter 4.3.2.3 --- Rolipram and Quazinone --- p.149 / Chapter 4.3.2.4 --- Ro 20-1724 and Quazinone --- p.150 / Chapter 4.3.3 --- Effects of the Sequential Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.150 / Chapter 4.3.3.1 --- Rolipram and Siguazodan --- p.150 / Chapter 4.3.3.2 --- Ro 20-1724 and Siguazodan --- p.151 / Chapter 4.3.3.3 --- Rolipram and Quazinone --- p.151 / Chapter 4.3.3.4 --- Ro 20-1724 and Quazinone --- p.152 / Chapter 4.4 --- Discussion --- p.153 / Chapter 5. --- Future Directions --- p.191 / Chapter 5.1 --- Future Directions --- p.192 / References --- p.195

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 113-137).