Pharmacology and Therapeutics - Theses
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ItemCharacterization and stress induced modulations of RXFP3+ neurons in the mouse BNST( 2017)Stress induced relapse and stressful emotional responses occurring during drug withdrawal are common problems for drug addicts. The relaxin-3/relaxin family peptide receptor 3 (RXFP3) system modulates stress responses and reward seeking behaviour. RXFP3 is expressed within the bed nucleus of the stria terminalis (BNST), and bilateral intra-BNST injections of an RXFP3 antagonist decreased self-administration and stress induced reinstatement of alcohol-seeking in rats. The electrophysiological properties of RXFP3+ neurons in the BNST and whether they are responsive to stress are presently not known. Similarly, the possibility of convergent signalling with other neuropeptides such as corticotropin releasing factor (CRF) in the BNST is not clear either. Therefore, whole cell recordings were conducted on defined RXFP3+ neurons in brain slices taken from adult RXFP3-Cre x tdTomato mice (n=38 mice for naive group). To study any alterations in RXFP3+ dorsal BNST neurons induced by chronic stress, a group of mice (n=13) were subjected to swim stress daily for 5 days. Mice were anaesthetised (5% isoflurane), decapitated and 250 µm coronal slices through the BNST were obtained. Based on Hammack’s characterization, the majority of recorded RXFP3+ neurons (~75%) were classified as Type II neurons and a new type of neuron, Type IV neurons were found within the dorsal BNST. Surprisingly, the electrophysiological profile of RXFP3+ neurons was not fixed; five days of swim stress induced plasticity, shifting a population of Type II neurons to Type III or Type IV neurons. Notably, stress induced plasticity of BNST RXFP3+ neurons was not only found in terms of neuron types, but also in their responsiveness to afferent inputs. The decay time of both spontaneous inhibitory postsynaptic currents (P=0.0005, Mann-Whitney test) and spontaneous excitatory postsynaptic currents (P=0.0028, Mann-Whitney test) were significantly increased after stress (naive, n=70 cells from 38 mice; stress, n=32 cells from 13 mice). Most BNST RXFP3+ neurons from the naive group (6 out of 9) were hyperpolarised following bath application of a selective RXFP3 agonist (A2, 100 nM), in line with an inhibitory role of this neuropeptide. Bath application of CRF (100 or 300 nM) onto brain slices during gap free voltage clamp recordings were conducted (n=14) to examine potential interactions between these peptide systems. CRF induced an increase of GABA release onto RXFP3+ neurons (~40% of the RXFP3+ neurons recorded), but decreased GABA release for ~35% of RXFP3+ neurons. In addition, CRF also caused direct postsynaptic effects on RXFP3+ neurons. Overall, these experiments have helped to elucidate the electrophysiological properties of RXFP3+ BNST cells, their responsiveness to stress, their interaction with a prototypic stress neuropeptide and their potential role in stress induced reward-seeking.
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ItemThe impact of cyclic AMP elevating agents on steroid resistance( 2016)Glucocorticoids are the most effective treatment for long-term control of asthma. However, a subset of patients require a much higher dose of glucocorticoids to achieve the effect of of treatment, and even the ceiling glucocorticoid response in those patients is lower. The insensitivity to glucocorticoid is termed glucocorticoid resistance. Many factors have been discovered to impair glucocorticoid response, including transforming growth factor beta (TGF-β). The research aimed to elucidate the effect of cAMP-elevating agents on TGF-β impaired glucocorticoid response. The experiments were mostly conducted in cells. GRE-SEAP assay was used to measure agonist activity and real-time polymerase chain reaction was used to measure the expression level of related genes. The results showed that compounds which induced higher cAMP level enhanced glucocorticoid response; whereas the repression caused by TGF-β and whether the repression can be attenuated by cAMP-elevating agents varied between genes and cell types. We attempted to investigate the effect of cAMP-elevating agents on glucocorticoid receptor (GR) translocation in airway epithelial cells, but GR predominantly distributed in the nucleus at the basal level and thus how cAMP influence GR translocation was not confirmed yet. A pilot study of glucocorticoid resistance in human nasal polyps is also included.
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ItemFunctional selectivity and kinetic features of orexin receptor antagonists( 2016)Orexin receptor antagonists are under development for the treatment of insomnia and a number of other diseases. However, the medium to high throughput screening procedures used to discover or characterize orexin receptor antagonists (and for that matter ligands targeting a variety of potential drug targets), frequently ignore two aspects of the new drugs candidates: their possible functional selectivity and genuine kinetic properties. Here, we studied several clinically effective and tool orexin receptor antagonists, which have been or are about to be evaluated in clinical trials, in several signaling pathways with an emphasis on kinetics. We investigated calcium mobilization and pERK elevation triggered by OXA in HEK293 cells stably transfected with OX1R or OX2R. We confirmed that the proposed antagonists behave indeed as antagonists in either assay. Most of them do not show significant functional selectivity between the two pathways, except for MK-1064, which inhibits calcium mobilization 35.5 times more potently than inhibiting pERK elevation. We also estimated the kinetic properties of the antagonists in radioligand competition, calcium mobilization and pERK assays. The results of radioligand competition and calcium mobilization assays consistently indicate that most tested antagonists dissociate from either orexin receptor slowly or extremely slowly, implying that equilibrium may be reached after several hours rather than a few minutes. Thus, SB-649868 is a very slow binder at OX1R as is almorexant at OX2R in both binding and calcium assays. However, results of the pERK assay lead to somewhat different conclusions: all tested antagonists (except almorexant at OX2R) seem to equilibrate with both orexin receptors relatively rapidly. The different conclusions drawn from the various assays may relate to experimental design, with different temperature and buffer conditions: some assays need to be performed with live cells at 37 degree, while others are performed for practical reasons at room temperature with cells or cell membranes. Orexin receptor ligands may have entirely different effects in different brain cells / nuclei if biased signaling was to occur. Independently of biased signaling, the kinetic properties of orexin receptor ligands may/will influence the actual receptor selectivity and duration of action of the compound, as is strongly suggested for clinically relevant antagonists such as almorexant, SB-649868, filorexant or suvorexant. Thus, this thesis stresses the importance of studying orexin receptor ‘antagonists’ in different functional assays and to investigate their kinetic properties under conditions that are as ‘physiological’ as feasible.
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ItemThe pharmacology of endothelin-1 in human and animal pulmonary and systemic vasculature( 2013)Endothelin-1 is a potent vasoactive peptide and has been suggested to play a major role in the pathogenesis of pulmonary hypertension, a debilitating disease characterised by increased pulmonary artery resistance and vascular remodelling. Current treatments for pulmonary hypertension include endothelin receptor antagonists and vasodilator agents, but these are unable to prevent disease progression and often have significant adverse systemic effects. The aims of this study were to investigate the effects of endothelin-1 on the pulmonary circulation in vitro to determine the involvement of nitric oxide, prostanoid mediators, calcitonin gene-related peptide and Ca2+ channels. Further, the efficacy of bosentan, an endothelin receptor antagonist currently used in the treatment of pulmonary hypertension, was compared to two novel antagonists, macitentan and ambrisentan. In addition, these studies aimed to investigate the effects of a number of vasodilator and vasoconstrictor agents in the pulmonary circulation and their responses were compared to those from systemic arteries to establish whether any pulmonary selectivity exists. Isolated pulmonary and systemic vessels from a number of animal species and humans were utilised in these studies. Concentration-response curves to endothelin-1 (10 pM – 300 nM) or sarafotoxin S6c (ETB receptor agonist; 10 pM – 300 nM) were constructed in isolated vessels in the presence or absence of antagonist treatments. In studies investigating relaxation responses, vessels were pre-contracted (to 65-80% maximum) with endothelin-1 or U46619 before being exposed to vasodilator agents. Endothelin-1 caused potent concentration-dependent contraction in all the tested vessels. In pulmonary arteries, endothelin-1 caused insignificant generation of nitric oxide, but substantial production of prostanoid mediators and these responses occurred independently of influx of extracellular Ca2+. Rat and rabbit interlobar pulmonary arteries were insensitive to the three tested endothelin receptor antagonists, however they effectively attenuated endothelin-1-induced contraction of human pulmonary and radial arteries and rat mesenteric arteries. Bosentan was able to antagonise ETB receptor-mediated contractions of rat interlobar pulmonary arteries, however it was at high concentrations (100 μM), suggesting it is approximately 50-fold less selective for ETB than ETA receptors. The vasodilator agents sildenafil, milrinone and BAY 41-8543 caused relaxation of pre-contracted rat pulmonary and mesenteric arteries, however no pulmonary selectivity was observed. Sympathomimetic vasopressor agents contracted both human pulmonary and radial arteries equipotently. However, whilst vasopressin caused potent contraction of radial arteries, it had no effect on human pulmonary arteries. In conclusion, regional differences in the responses to endothelin-1 and sarafotoxin S6c were evident between large and small pulmonary arteries reflecting receptor heterogeneity in the pulmonary circulation. The pKB values calculated for bosentan suggest it is approximately 50-fold more selective for ETA than ETB receptors. As a result, the classification of bosentan as a ‘dual’ receptor antagonist is improper and it should be considered ETA receptor-selective. The novel vasodilator agent BAY 41-8543 effectively attenuated endothelin-1-mediated contractions of rat interlobar pulmonary arteries and may present possible new therapeutic target for pulmonary hypertension, either alone or in combination with an endothelin receptor antagonist.
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ItemCharacterization of TRPV4 in inherited disorders( 2012)The Transient Receptor Potential Vanilloid 4 channel (TRPV4) is a Ca2+ permeable, nonselective cation channel that is important for sensation of several physical and chemical stimuli and also involved in multiple physiological functions. Recently it gained immense scientific and clinical interest as several independent studies demonstrated that TRPV4 mutations cause heritable genetic disorders including several skeletal dysplasias and peripheral neuropathies. TRPV4 thus plays a critical role in skeletal and neurological development. Our recent gene linkage studies on a novel skeletal disorder called Familial Digital Arthropathy-Brachydactyly (FDAB) identified mutations in TRPV4. The main objective of the present study was to comprehensively understand the effects of FDAB mutations on channel function. To facilitate this aim, stable cell lines expressing TRPV4 wild type and mutants were generated. FDAB mutants expressed in HEK293 cells showed a defect in maturation from high mannose to complex glycosylation. Our data also show that constitutive channel activity was increased in cells expressing mutant ion channels compared to wild type TRPV4. Responses to the synthetic agonists, GSK1016790A and 4αPDD, were reduced, reflecting less cell surface expression of TRPV4. In addition, responses to hypotonicity were significantly reduced, suggesting a disruption of the normal mechanism of hypotonic activation of TRPV4. Close analysis of the data obtained from these naturally occurring genetic mutants as well as other TRPV4 mutants suggest that it is not the altered channel activity of these mutants per se, but the involvement and interaction of other factors that seem to modulate oligomerization, trafficking and degradation of TRPV4 channels. TRPV4 functions not alone but as part of a multiprotein signaling complex that is critical for mechanosensation, pain, hyperalgesia and other physiological functions. Signaling complexes are an evolutionary adaptation that enhance the sensitivity and efficiency of signaling between sensors and effectors (e.g., ion channels), and which confine signaling events to precise subcellular locations. The study was also designed to define key components of the TRPV4 signaling complex and to examine the hypothesis that disruption of components of the signaling complex will affect channel function. A receptor for activated protein kinase C (RACK1) was identified as a novel candidate signaling complex component, however, the role of RACK1 in TRPV4 function still needs to be verified experimentally. In summary, this work identifies TRPV4 mutations that lead to reduced channel activation as a new mechanism underlying an inherited osteoarthropathy and raises the possibility that modulating TRPV4 activity could provide a new therapeutic approach for osteoarthritis. The identification of RACK1 as a component of the TRPV4 protein complex validates our hypothesis that TRPV4 exists in a signaling complex and also presents insights into novel therapeutic strategies for pain syndromes.