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Hv VITRO MODEL of S'i'roke in the RAT HPPOCAMPUS

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Hv VITRO MODEL of S'i'roke in the RAT HPPOCAMPUS NOVEL ORGANIC NlTRATES AS POSSIBLE NEUROPROTECTANTS IN AN hV VITRO MODEL OF S'I'ROKE IN THE RAT HPPOCAMPUS Allison Elizabeth Clarke A thesis submitted to the Department of Pharmacology and Toxicology in conformity with the requirements for the degree of Master of Science Queen's University Kingston, Ontario, Canada May, 2001 Copyright O Allison Elizabeth Clarke, 2001 National Library Bibliothèque nationale ($1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 WeUingîon Street 395, nie Wellington Ottawa ON KIA ON4 OttawaON K1AON4 Canada canada The author has granted a non- L'auteur a accordé une Licence non exclusive licence dowing the exclusive permettant à la National Libraiy of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The auîhor retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Allison Elizabeth Clarke: Novel Organic Nitrates as Possible Neuroprotectants in an In Vitro Mode1 of Stroke in the Rat Hippocampus. M.Sc. Thesis, Queen's University, Kingston, Ontario, Canada, May, 2001. Novel organic nitrates are a group of established nitric oxide donors based on the chemical structure of glyceryl trinitrite (GTN). It has been previously suggested that nitnc oxide can potentially play a neuroprotective role in ischemia due to its ability to: inhibit ~a'+influx through the N-methyl-D-aspartate (NMDA) receptor, act as an antioxidant and increase cGMP levels in the neuron. A group of investigators examining the neuroprotective properties of the secreted fom of amyloid precursor protein (sAPPu) have discovered a protein kinase G (PKG)dependent. cGMP mediated mechanism. They have postulated that this neuroprotection is due to: activation of K' channels, inhibition of the NMDA receptor and enhancement of glucose and glutamate uptake into synaptic compartrnents. This thesis tested the hypothesis that novel organic nitrates are neuroprotective in an in vitro model of stroke possibly due to a cGMP mediated mechanism. The first objective was to establish the in vitro model of stroke with respect to testing of known neuroprotectants such as hypothermia and determining an appropriate length of insult. A half hour insult time was chosen because it caused a subrnavimal increase in lactate dehydrogenase (LDH) release. LDH release was used as a marker of ce11 viability. The induction of hypothermia during the ischemic insult completely protected the hippocarnpal slices from the in vitro iscliemic insult. The in vitro ischemic insult involved low[Oz] and low[glucose] in the incubation buffer. The second objective was to determine if the novel organic nitrates had any neuroprotective properties and if any observed neuroprotection was dependent upon cGMP generation. Three novel organic nitrates, GT-091, GT-094 and GT-310 were significantly protective against low[02] and low[glucose]. Similarly, the cGMP analogue, dibutyql cGMP was also neuroprotective in the same model suggesting that the neuroprotection observed with the novel organic nitrates may be due to a cGMP mediated mechanism. Unexpectantly, the neuroprotection provided by GT-094 could not be attenuated by CO-application of 1H- [ 1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. Additionally, GT-094 was unable to increase cGMP levels in hippocampal slices der hypoxialhypoglycemia as assessed by a cGMF radioirnrnunoassay. These results indicate that the neuroprotective mechanism of GT-094 does not involve cGMP generation. Interestingly, CAMP could mimic the neuroprotection observed with cGMP. A protein kinase A (PU) or PKG inhibitor could not attenuate the neuroprotective effects of CAMP and cGMP, respectively. In summary, these findings suggest GT-094 and the cyclic nucleotides are exerting neuroprotection by two separate and independent mechanisms. Furthemore, these results indicate that the cyclic nucleotides are acting by a pathway that does not involve PKA or PKG activation. 1 would like to take this opportunity to thank my supervisor Dr. Roland J. Boegman for al1 his guidance and insight in the completion of this thesis project. 1 would also like to thank Dr. James N. Reynolds for al1 his invaluable help. 1would like to acknowledge and thank Lihua Xue and Diane Andenon for generously providing me with some of the data contained in this thesis. Their help is greatly appreciated. Lihua Xue completed the LDH study on Sin4 chloride CO-administeredwith ODQ as well as the cresyl violet staining with the hippocampal slices treated with Sin-1 chlonde and GT-094. Diane Anderson provided me with the RIA data on GT-094 and ODQ. 1 would also like to thank the other members of the GoBang team: Dr. Jhamandas. Dr. Bennett, Dr. Thatcher, Margo Poklewska-Koziell and Adrian Nicolescu. GoBang Therapeutics and Queen's Medical Discoveries primarily financed this research. Queen's School of Graduate Studies provided personal funding. This thesis is dedicated to my farnily. TABLE OF CONTENTS Page.. Abstract 11 S.. Acknowledgrnents 111 Table of Contents v List of Figures vii List of Abbreviations and Symbols ix 1 INTRODUCTION 1 1.1 S tatement of Research Problem 1 1.2 Ischemic Ce11 Damage 3 1.2.1 NMDA Receptor Antagonists 5 1.2.2 Metabotropic Glutamate Receptors 7 1.3 Nitric Oxide 8 1.3.1 Nitric Oxide Synthase Antagonists 10 1.3.2 Nitric Oxide and Neurotoxicity 11 1 -3.3 Peroxynitrite 12 1.3.4 Nitrk Oxide Production and Neuronal Outcome 12 1.3.5 Nitric Oxide and Neuroprotection 12 1.3.6 Niûic Oxide and NMDA Receptor Inhibition 14 Antioxidant Properties of Nitric Oxide 15 18 cGMP and Neurotoxicity 18 cGMP and Neuroprotection 19 cGMP and P-Arnyloid Precursor Protein 20 Glutamate and Glucose Uptake 21 Inhibition of the NMDA Receptor 2 1 Potassium Channels 22 5 Cyclic P{ucleotide Gated Ion ChanneIs 23 1.6 Phosphodiesterases and Ischemia 23 1.7 CAMP 25 1.8 Guanine Nucleotide Exchange Factors 26 1.9 Novel Organic Nitrates 27 1.10 In Vitro Mode1 of kchernia 29 1.1 1 Research Rationale, Hypothesis and Objectives 2 METHODS AND MATERIALS 2.1 Chemical Solutions 2.2 Experimental Animals 2.3 Tissue Isolation 2.4 LDH Assay 2.5 Protein Determination 2.6 cGMP Radioimmunoassay 2.7 Cresyl Violet Staining 2.8 Data Analysis 3 RESULTS 3.1 Time Course of LDH Release 3.2 Temperature and LDH Release 3.3 Conventional NO Donors 3.4 Novel Organic Nitrates 3.5 Synthetic cGMP Analogues 3 S.1 Dibutyryl cGMP and Rp-8-pCPT-cGMP 3.6 Synthetic CAMP Analogues 3.6.1 Dibutyryl CAMPand H-89 3.7 Dibutyiyl CAMP and Forskolin 3.8 ODQ 3.9 cGMP Radioirnmunoassay 3.10 Cresyl Violet Staining 4 DISCUSSION 4.1 Future Research Directions Re ferences Vita LIST OF FIGURES Page 1.1 Sumrnary of hypoxic/hypoglycemic injury 6 1.2 A schematic depicting NO production by NOS 1.3 Summary of the neurotoxic properties of NO 1.4 Sumrnary of the neuroprotective properties of NO 1.5 Chemical structure and proposed biotransformation of the novel organic nitrates 3.1 Rat hippocarnpal slices exposed to differing lengths of hypoxiahypoglycemia 3.2 Rat hippocampal slices exposed to hypothermie conditions 3.3 Rat hippocampal slices treated with Sin-1 chlonde 3.4 Rat hippocarnpal slices treated with NO-exhausted Sin-1 chloride 3.5 Rat hippocarnpal slices treated with GSNO 3.6 Rat hippocampal slices treated with GT-09 1 3.7 Rat hippocampal slices treated with GT-094 3.8 Rat hippocampal slices treated with GT-3 10 3.9 Hippocarnpal slices treated with 1mM 8-bromo-cGMP 3.10 Rat hippocarnpal slices treated with dibutyryl cGMP 3.1 1 Rat hippocampal slices treated with cGMP 3.12 Rat hippocarnpal slices treated with 8-pCPT-cGMP 3.13 Rat hippocampal slices treated with dibutyryl cGMP and Rp-8-pCPT-cGMP 3.14 Rat hippocarnpal slices treated with dibutyryl CAMP 3.15 Rat hippocampal slices treated with I mM 8-bromo-c AMP 3.16 Rat hippocampal slices treated with dibutyryI CAMP and H-89 3.1 7 Rat hippocampal slices treated with 50pM fonkolin and 100pM dibutyryl cGMP 3.1 8 Rat hippocampal slices treated with 50pM GT-094 and 0.5pM ODQ 3.19 Rat hippocampal slices treated with Sin- 1 chloride and ODQ 3.20 Rat hippocarnpal slices treated with ODQ 3.2 1 Concentration of cGMP in rat hippocampal slices 3.22 Rat hippocampal slices stained with cresyl violet 4.1 Proposed mechanism of action of the novel organic nitrates 4.2 Proposed mechanism of neuroprotection mediated by cGMP LIST OF ABBREVIATIONS AND SYMBOLS a alpha AP arnyloid beta peptide AMPA a-arnino-3-hydroxy-5-methyl-4-isoxazole propionate ANOVA analysis of variance P beta BSA bovine semalbumin ca2+ calcium ion CaM calmodulin CaMK II calcium-calmodulin-dependent protein kinase II CAMP cyclic adenosine 3'5 '-monophosphate CBF cerebral blood flow cGMP cyclic guanosine 3 ' 5 '-monophosphate CNS centnl nervous system CPT CAMP 8-(4-c hlorop heny1thio)-adenosine 3 ' :5 ' -cyclic- monophosphate CREB cyclic AMP-responsive element binding protein DHPG 3'5-dihydroxyphenylglycine DMSO dimethyl sulfoxide eNOS endothelial nitric oxide Epac exchange protein directly activated by cyclic AMP FAD flavin
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