Orientador: Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T08:00:03Z (GMT). No. of bitstreams: 1 Ramos-Filho_AntonioCelsoSaragossa_D.pdf: 3024613 bytes, checksum: a03a80c65d863acd441249f461461216 (MD5) Previous issue date: 2014 / Resumo: Os receptores TRPV4 e TRPM8 são expressos no urotélio e em fibras aferentes sensitivas da bexiga. Fisiologicamente, a ativação mecânica do receptor TRPV4 na parede da bexiga participa do controle miccional. Em doenças de origem inflamatória, esses receptores adquirem funcionalidade importante. As disfunções da bexiga no diabetes podem estar associadas a alterações ao nível de detrusor, inervação e urotélio. A disfunção urotelial parece ser a responsável por desencadear as alterações neurais e musculares da bexiga. Assim, o objetivo do presente estudo foi investigar os mecanismos fisiopatológicos da ativação dos receptores TRPV4 e TRPM8 no estado diabético em camundongos. Para tanto, dividimos o estudo em duas etapas, sendo que na primeira avaliamos a participação dos receptores TRPV4 e TRPM8 nos mecanismos contráteis e relaxantes do detrusor isolado de animais controles e knockout para esses canais. Em uma segunda etapa estudamos a ativação desses canais em camundongos diabéticos pela injeção intraperitoneal de estreptozotocina (180 mg/Kg) por 4 semanas. Em fragmentos do detrusor isolados de camundongos mostramos que o agonista do receptor TRPV4, GSK1016790A, causou resposta contrátil dependente da concentração. Por outro lado, quando os tecidos foram contraídos com solução despolarizante de KCl, o GSK1016790A causou relaxamento da preparação. No detrusor isolado de animais TRPV4-/- verificamos hipercontratilidade ao carbacol (agonista muscarínico) e à estimulação elétrica, assim como redução no relaxamento ao agonista ?-adrenérgico não-seletivo, isoprenalina. Estes efeitos não foram obtidos com os antagonistas dos receptores TRPV4, RN1734 e HC067047. A indução do diabetes causou nocicepção mecânica e aumento da proporção entre bexiga e peso corpóreo após 4 semanas da injeção. A avaliação miccional dos animais diabéticos mostrou aumento da capacidade, frequência urinária e das contrações involuntárias da bexiga. Observamos ainda hipercontratilidade do detrusor ao carbacol, à estimulação elétrica e ao KCl. A indução do diabetes em animais TRPV4-/- não modificou as disfunções "in vivo" e "in vitro" observadas nos animais wyld type diabéticos, mostrando que a ausência crônica dos receptores TRPV4 desencadeia alterações miccionais que são anteriores as causadas pelo diabetes. Também verificamos que os animais TRPM8-/- não apresentam alteração na resposta contrátil ao carbacol e à estimulação elétrica. Por outro lado, o mentol, mas não a icilina, reduziu significativamente as respostas contráteis nestes animais. O mentol inibiu o influxo de cálcio extracelular em cultura de células da musculatura lisa da bexiga por mecanismo inibitório direto nos canais Cav1.2. O tratamento agudo com mentol, intraperitoneal e intravesical, atenuou as disfunções miccionais observadas nos camundongos diabéticos. "In vitro" o pré-tratamento com mentol reduziu a hipercontratilidade ao carbacol no grupo diabético, sem alterar a resposta no grupo controle. Concluímos que o mentol impede a resposta contrátil da bexiga por mecanismo independente do receptor TRPM8 bloqueando o influxo de cálcio extracelular nos canais Cav1,2, podendo ser utilizado como tratamento na hiperatividade de bexiga de origem miogênica / Abstract: The TRPV4 and TRPM8 receptors are expressed in bladder urothelium and sensitive afferent fibers. Physiologically, the mechanical activation of TRPV4 receptor in the bladder wall is involved in micturition control. In inflammatory diseases, these receptors may have important roles. The bladder dysfunction in diabetes may be associated with changes at the level of detrusor, innervation and urothelium. The urothelial dysfunction triggers neural changes, modifying consequently the smooth muscle contractility. Thus, the goal of the present study was to investigate the pathophysiological mechanisms of TRPV4 and TRPM8 receptor activation in physiological and diabetic conditions in mice. For this purpose we divided the study in two phases, the first of which we evaluated the participation of TRPV4 and TRPM8 receptors in detrusor contractile and relaxing mechanisms in control and knockout animals for these channels. In the second phase we studied the activation of these channels in diabetic mice induced by intraperitoneal injection of streptozotocin (STZ; 180 mg / kg, 4 weeks). The TRPV4 agonist GSK1016790A produced concentration-dependent detrusor contractions. On the other hand, in detrusor pré-contracted with KCl (80 mM), GSK1016790A caused relaxation responses. In TRPV4-/- animals, we verified hypercontractility to carbachol (muscarinic agonist) and electrical-field stimulation, as well as a decreased relaxation to isoprenaline (non-selective ?-adrenergic agonist). These effects were not obtained with the TRPV4 antagonists, RN1734 and HC067047. Induction of diabetes with STZ caused hyperglycemia, mechanical nocicepton, and increased ratio between bladder and body weight after 4 weeks. The miccturition evaluationin diabetic animals showed increased capacity, urinary frequency, and non-voiding contractions. Hypercontractility to carbachol, electrical-field stimulation and KCl in isolated detrusor were lso observed. The induction of diabetes in TRPV4-/- animals did not change the urinary dysfunctions. Our data are consistent with the proposal that TRPV4 receptor has a physiological function in micturition control by decreasing muscarinic-induced contractions and increasing ?-adrenergic-mediated relaxations. Moreover, the bladder contractions to carbachol and EFS in TRPM8-/- did not significantly change compared to TRPM8+/+. However, menthol (300 ?M), but not icilin (1 ?M), significantly inhibited these contractile responses. The menthol (300 ?M) inhibited extracellular calcium influx in bladder smooth muscle cell culture by direct mechanism though Cav1.2 channels. In addition the acute treatment with menthol, intraperitoneal and intravesical, atenuated the micturition dysfunctions observed in diabetic mice. Also, detrusor preparations pre-treated with menthol decreased carbachol hypercontractility, without changing the responses in normoglycemic group. Menthol reduces bladder contractions by mechanisms independent of TRPM8 receptor activation, inhibiting extracellular calcium influx through Cav1.2 channel, thus been considered as treatment for bladder overactivity of myogenic origin / Doutorado / Farmacologia / Doutor em Farmacologia

Orientador: Elaine Minatel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:31:19Z (GMT). No. of bitstreams: 1 Burgos_RafaelRamosde_M.pdf: 2000317 bytes, checksum: 3ec9bdb6204407b72a6bd5378a5656ae (MD5) Previous issue date: 2013 / Resumo: Tratamentos com antioxidantes e bloqueadores de canais de cálcio têm apresentado resultados positivos na diminuição da mionecrose nas fibras musculares distróficas. O objetivo do presente projeto foi verificar se o tratamento com o bloqueador de cálcio Nifedipina (Ni) e o antioxidante Coenzima Q10 (CoQ10), administrados isoladamente ou em associação antes que se iniciem os ciclos de degeneração/regeneração muscular, pode apresentar efeito benéfico sobre as fibras musculares distróficas de camundongos mdx, modelo experimental da distrofia muscular de Duchenne. Camundongos mdx com 14 dias de vida pós-natal foram divididos em 4 grupos experimentais: (1) tratados com álcool 20% e solução aquosa 1% de Tween 80; (2) tratados com a associação de Ni e CoQ10; (3) tratados com Ni isoladamente; (4) e tratados com somente CoQ10. Camundongos da linhagem C57BL/10 foram usados como controle. Os músculos tibial anterior (TA), diafragma (DIA) e esternomastóideo (STN) foram retirados e utilizados para quantificar: (1) o número de fibras em degeneração, regeneradas e normais; (2) o nível de TNF-?, NF-?B e 4-HNE; (3) o conteúdo de cálcio total; e (4) a área de inflamação muscular. Amostras de sangue foram utilizadas para determinação dos níveis creatina quinase (CK). Os animais tratados com Ni apresentaram: redução significativa de fibras em degeneração no músculo TA, redução nos níveis de TNF-? em todos os músculos analisados e no conteúdo de NF-?B e cálcio no músculo DIA. Animais mdx tratados com Coenzima Q10 mostrou redução: CK, TNF-? no músculo TA, no conteúdo de NF-?B nos músculos DIA e STN, na área de inflamação no músculo DIA e determinação do cálcio nos músculos DIA e STN. A associação dos tratamentos demonstrou redução significativa na análise de CK, no TNF-? do músculo TA, na área de inflamação, conteúdo do NF-?B e cálcio no músculo DIA. O conjunto dos resultados sugere que a Ni e a CoQ10 possam ser potencialmente úteis para o tratamento farmacológico da distrofinopatias. Entretanto, estudos futuros da dosagem e do tempo de tratamento serão necessários para obtenção de efeitos benéficos mais significativos sobre os músculos distróficos / Abstract: Treatment using antioxidant and calcium channel blockers have shown positive results for myonecrosis decrease in dystrophic muscle fibers. The aim of this study is to verify if treatment with Nifedipine (Ni) calcium channel blocker and Coenzyme Q10 (CoQ10) antioxidant, administered alone or in association before degeneration/regeneration cycles take place, may have a beneficial effect on dystrophic muscle fibers in mdx mice, which are the experimental model for Duchenne's dystrophy. Mdx mice at 14 days old were divided into 4 experimental groups: (1) treated with 20% alcohol and 1% Tween 80 water solution; (2) treated with an association of Ni and CoQ10; (3) treated with Ni alone; and (4) treated with CoQ10 alone. C57BL/10 mice were used as a control. The tibialis anterior (TA), diaphragm (DIA) and sternomastoid (STN) muscles were removed and used for quantification of: (1) number of degenerated, regenerated and wild-type fibers; (2) TNF-?, NF-?B and 4-HNE levels; (3) total amount of calcium; and (4) muscle inflammation area. Creatine kinase (CK) was analyzed from a blood sample. The animals treated with Ni showed: a significant decrease of degenerated muscle fibers in the TA muscle, a reduced TNF-? level in all muscles analyzed and lower levels of NF-?B and calcium in the DIA muscle. Mdx mice treated with Coenzyme Q10 presented a decrease of the following: CK expression, TNF-? level in the TA muscle, NF-?B level in the DIA and STN muscles, inflammation area in the DIA muscle and calcium content in the DIA and STN muscles. The association of both drugs showed a significant reduction of CK in the blood, TNF-? in the TA muscle, and also a reduction of the inflammation area, the NF-?B expression and the calcium content of the DIA muscle. Overall results suggest that Ni and CoQ10 may play a potential role as a pharmacological treatment for dystrophynopathies. However, further studies must be carried out for both dosage and treatment period to obtain more significant beneficial effects on dystrophic muscles / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Nouvelles voies de modulation des canaux calciques de type T.Grâce à leur rôle dans l'excitabilité cellulaire et l'homéostasie calcique, les canaux calciques de type T participent à différentes fonctions physiologiques telles que le sommeil ou le contrôle du rythme cardiaque et de la pression artérielle. Ils sont également impliqués dans certaines pathologies comme la douleur ou l'épilepsie. La régulation de l'activité des canaux de type T est encore mal connue et c'est l'enjeu de cette thèse. Dans une première partie, nous avons caractérisé l'effet de certains lipides endogènes sur ces canaux, en particulier les métabolites de l'acide arachidonique, et identifié le 5,6-EET comme un nouveau bloqueur des canaux de type T. Nous avons ensuite évalué l'existence d'un site de liaison des lipoamino acides sur les canaux de type T à l'aide d' d'expériences de compétitions réalisées avec un inhibiteur spécifique de ces canaux, la molécule TTA-A1.Dans une deuxième partie, nous avons étudié la régulation par le calcium des canaux de type T. Nous avons montré que l'entrée de calcium par les canaux T eux-mêmes ou par activation des récepteurs P2X4 et NMDA induisait une inhibition du courant de type T. Le calcium activerait une phosphatase qui provoquerait un déplacement de la courbe d'inactivation à l'état stable vers les potentiels négatifs, réduisant ainsi la disponibilité des canaux. Ce phénomène d'inhibition du courant lié à l'entrée du calcium pourrait être un mécanisme de rétrocontrôle négatif limitant l'entrée de calcium dans la cellule afin d'éviter une toxicité provoquée par une concentration de calcium intracellulaire trop élevée. Suite à cette inhibition, lorsque l'entrée de calcium est arrêtée, le courant augmente. Cette augmentation semble être due à l'intervention de la protéine kinase Pak qui rendrait les canaux de type T disponibles.En conclusion, nous avons identifié et caractérisé deux nouveaux mécanismes endogènes de régulation des canaux de type T : une modulation par les lipides, et une modulation par les calcium et la protéine kinase Pak. / New pathways of regulation of T-type calcium channels.Thanks to their role in cellular excitability and calcium homeostasis, T-type calcium channels are involved in several physiological functions such as sleep or control of cardiac rythmicity and vascular tone. They are also involved in some diseases such as pain or epilepsy. The regulation of T-type calcium channels is still poorly understood and that is the challenge of this thesis.In a first part of the study, we caracterised the effect of some endogenous lipids on these channels, particularly the metabolites of arachidonic acid, and identified the 5,6-EET as a new blocker of T-type channels. We then evaluated the existence of a binding site of lipoamino acids on T-type channels using binding experiments made with a specific inhibitor of these channels, the TTA-A1 molecule.In a second part, we studied the regulation of T-type channels by calcium. We showed that a calcium entry through T-type channels or through P2X4 and NMDA receptors activation induced an inhibition of T-type current. Calcium would activate a phosphatase which would trigger a shift of the steady-state inactivation curve toward negative potentials, reducing the availability of channels. This phenomenon of current inhibition due to calcium entry may be a feedback mechanism limiting calcium entry in the cell to avoid toxicity due to a too high intracellular calcium concentration. After this inhibition, when calcium entry is stopped, the current increases. This increase seems to be due to the intervention of the Pak protein kinase which would make T-type channels available again.In conclusion, we studied and caracterised tow new mechanisms of T-type channels regulation: a modulation by lipids, and a modulation by calcium and the Pak protein kinase.

Voltage-gated Ca2+ (Cav) channels are activated upon depolarization. They specifically allow Ca2+ ions to come into the cell. These Ca2+ ions are bi-functional because they not only control cell excitability but also couple electrical activity to complex downstream signaling events, such as excitation-contraction coupling in muscles and neurotransmitter release in neurons. In the brain, Cav channels are expressed in the pre- or post-synaptic membrane of most excitable cells, neurons. In the past few years, their expression and function have also been characterized in many nonexcitable cells such as astrocytes. This dissertation focuses on the regulation of one subtype of postsynaptic Cav channels, Cav1.2, in neurons. In the first part of chapter I, I provide a literature overview of Cav channels in terms of their subtypes, localizations, physiological functions, and biophysical properties. For years, Cav channels were studied as single entities. But now, based on multiple proteomic studies, we know that these channels actually do not live alone. They interact with numerous proteins depending on the physiological conditions. Such interactions can anchor the channels to optimal sites of action, and tether Cav channels to their modulatory molecules. Therefore, it is crucial to understand how Cav channels are regulated by their macromolecular assembly. Among these protein partners, our lab studied the regulation of Cav channels by a subset of PDZ-domain containing proteins. Because these proteins play an important role in scaffolding and they colocalize with both pre- and post-synaptic Cav channels. Indeed, previous studies from our lab and other groups have revealed that PDZ proteins participate in a multitude of Cav regulation. The second part of chapter I introduces the diverse modulation of neuronal Cav channels by numerous PDZ proteins. In neurons, Cav1.2 channels regulate neuronal excitability and synaptic plasticity. Their functions have been implicated in learning, memory, and mood regulation. A study published in the journal Lancet showed that the gene encoding Cav1.2 is a common risk factor for five major psychiatric disorders. A PDZ protein, densin-180 (densin) is an excitatory synapse protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin exhibit similar behavioral phenotypes that closely match those in mice lacking Cav1.2. In chapter II and III, we investigated the functional impact of densin on Cav1.2 channels and their auxiliary subunit β2a. Besides the regulation of Cav channels by their interactome, we have also known for a long time that Ca2+ currents undergo a negative feedback regulation. This regulation is called Ca2+-dependent inactivation (CDI) and it is mediated by Ca2+ that directly traverses the pore. CDI has been described for Cav channels in multiple cell types. In the heart, CDI prevents excessively long cardiac action potentials, which in turn can prevent activity-dependent arrhythmia. In neurons, CDI may be neuroprotective by preventing excitotoxic Ca2+ overloads. In the last 18 years, two essential components have been revealed in the mechanism of CDI. One is the protein calmodulin (CaM). CaM interacts directly with sites on the C-terminus of Cav channels. It binds to the incoming Ca2+ ions and produces a mysterious conformational change that determines the conductance of the channel. The other molecular player is Cavβ protein family. Cavβ comprises four subfamilies β1 through β4, which generally enhance the channel inactivation, except β2a. In chapter IV, Xiaohan Wang from Roger Colbran’s lab in Vanderbilt University, and I identified a new molecular determinant for Cav1.2 CDI. The α2δ subunit is an extracellular component of the Cav channel complex. Similar to Cavβ subunits, α2δ subunits are essential for the biophysical properties, surface level, and trafficking of Cavα1 subunits. There are four isoforms of α2δ subunits (α2δ1 to α2δ4). They display distinct tissue distributions. Although the roles of α2δ subunits in Cav channel regulation were studied extensively, studies have proposed that the function of α2δ subunits may be in part or entirely independent of Cav channel complex, such as synaptogenesis. Considering the important role of α2δ in physiology and pathology, it is imperative to identify the factors that regulate the properties of α2δ. In chapter V, I explored the trafficking dynamics of α2δ1 and revealed a potential regulator of α2δ1 for its protein stability and localization. One beauty of doing research is that it always motivates us to think and ask more questions on our journey of demystifying nature. While looking at the evidence that I find, I realize how much more we could do in the future. In chapter VI, I conclude the findings of each chapter and share my perspectives on the future direction for these research projects.

Calcium (Ca2+) is a universal second messenger controlling a wide variety of cellular reactions and adaptive responses. All the versatility of a Ca2+ signaling requires that the concentration of Ca2+ ions in the cytoplasm be highly regulated. Generation of Ca2+ mobilizing signals in cells involves regulation by multiple components controlling Ca2+ release from the internal stores, Ca2+ influx across the plasma membrane, elicitation of Ca2+ sensitive processes and finally the removal of Ca2+ from the cells. Inositol-1, 4, 5-trisphosphate receptors (IP3Rs) and ryandine receptors (RyRs) are the most studied Ca2+ release channels located on the internal stores. Previous studies have shown ryanodine receptors (RyRs) play a key role in the process of Ca2+ signaling participating in the oscillatory patterns of controlling the release of Ca2+ from ER and regulating the influx of Ca2+ by coupling with plasma Ca2+ channels. Although recent progress deciphered the behavior and function of RyRs in regulation of Ca2+ signal, it still remains mysterious in understanding the molecular mechanism of its regulation and its connection with plasma membrane Ca2+ channels in neuronal cells. Here this study aimed to utilized the most cutting-edge RNA interference techniques, along with well-characterized pharmacological regulators of RyRs, to better characterized the role of RyRs is our neuronal cell line model NG115-401L. Our first main goal of this project was to develop an effective protocol that could selectively knockout or knockdown expression levels of the RyR1 gene in NG115-401L cells. After testing different siRNA primers including their combination with different transfection reagent, the result shows a significant silencing effect to the RyR1 mRNA expression levels. In the second part of this study, we used a group of pharmacological agents with well-known regulatory actions on RyRs to characterize the functional roles of the RyRs expressed in NG115-401L cells. All four agonists which are ryanodine, caffeine, CMC and PCB 95 show their abilities to activate the RyRs, increase [Ca2+]iand induce the influx of Ca2+ via SOC. After transfected NG115-401L cells by siRNA, the Ca2+ release and influx signals were highly diminished suggesting RyR1 gene was successfully knocked down and the successfully knocked down and the Ca2+ mobilization mediated by RyR1 was decreased greatly. Finally in order to study the effects of the regulation of Ca2+ by RyR modulators and RyR gene knockdown on cell growth patterns and cell viability, the NG115-401L cells were exposed to various concentrations of RyR regulators and siRyR1 primer for different time periods. The siRNA transfection showed the least effect on cell growth, as compared with pharmacological agents that modulate RyR function. Considering we achieved high levels of gene knockdown and its low cytotoxity, our result suggests that siRNA silencing for RyRs may become a promising gene therapeutic target in the future.

Background: The Wnt/β-catenin signalling pathway is activated in arrhythmogenic heart diseases such as myocardial infarction and heart failure, but it is unclear if the pathway regulates cardiac ion channels and thus may play a role in arrhythmogenesis. Previous PCR array screening from our lab showed that the transcript level of the T-type calcium channel gene Cacna1g was reduced in primary culture of neonatal rat ventricular myocytes (NRVMs) after activation of Wnt/β-catenin signalling with Wnt3a protein (100 ng/ml) or a small molecule activator of the pathway, CHIR (3 µM) (n=3, p<0.01). In this study, we examined the effects of Wnt/β-catenin signalling on T-type calcium channels (Caᴠ3.1), which play a key role in the pacemaker function of the sinoatrial node (SAN). Results: RT-qPCR and western blot demonstrated dose-dependent reductions in Cacna1g mRNA (n=7, p<0.01) and Cav3.1 protein (n=4, p<0.01) in NRVMs after treatment with CHIR (3 μM). There was also a decrease in Cacna1g mRNA in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after treatment with CHIR (5 μM) (n=4; p<0.001). Patch-clamp recording demonstrated reduced T-type calcium current (ICa,T) in NRVMs after Wnt3a treatment (3 μg/ml) (n=5, p<0.05). In isolated mouse SAN tissue, perfusion with an ICa,T blocker, ML-218 (30 µM), led to dose-dependent reductions in spontaneous beating rate (n=4, p<0.0001) indicating a critical role of ICa,T in SAN pacemaking. In adult rats, activation of Wnt/β-catenin signalling through the application of CHIR in a poloxamer gel to the SAN region did not alter the in vivo heart rate in electrocardiogram (ECG) (n=8, p=0.12). However, ex vivo culture of SAN tissue from the in vivo experiments revealed a reduction intrinsic beating rate in the CHIR treated group (n=7) compared to the control (DMSO) (n=8) (p<0.05). Summary: Wnt/β-catenin signalling inhibits T-type Ca²⁺ current in cardiomyocytes by, at least partly, reduced Cacna1g mRNA and Cav3.1 protein. Activation of Wnt/β-catenin signalling reduces the intrinsic heart rate likely by inhibition of T-type Ca²⁺ current in SAN pacemaker cells.

The purpose of this work was to determine whether the previously identified fatty acid activation of large conductance Ca2+-activated K+ (BK) channels from rabbit pulmonary artery smooth muscle cells was due to the direct interaction of the fatty acid with a site on the channel protein. If this was found to be the case, this study would also attempt to identify the site of fatty acid-protein interaction. Fatty acids released from membrane phospholipids by cellular phospholipases or available to the cell from the extracellular environment are important signaling molecules. Fatty acids can modulate the activity of a large number of molecules including protein kinases, phospholipases, adenylate and guanylate cyclases, G-proteins and ion channels. Fatty acids have also been shown to activate transcription of genes belonging to the steroid/thyroid superfamily of receptors. The actions of fatty acids on signal transduction pathways can be direct, whereby the fatty acid molecule itself is responsible for changes in the activity of enzymes, ion channels and other proteins. Alternatively, the effects of fatty acids may be indirect. In this case, biologically active lipids, produced from the metabolism of arachidonic acid are responsible for changes in cellular signaling. A previous study on the fatty acid modulation of rabbit pulmonary artery smooth muscle BK channel activity concluded that channel activation by fatty acids did not involve cycloxygenase, lip oxygenase and P450 metabolites (122), eliminating this indirect action of fatty acids as a possible mechanism. When dealing with the effects of fatty acids on membrane bound ion channel proteins, other mechanisms of action are also possible. For example, fatty acids are capable of entering the cell membrane and can thus affect properties of the lipid bilayer, such as membrane fluidity or membrane surface charge, that may consequently alter the activity of ion channel proteins. In addition, fatty acid mediated alterations of ion channel activity could result from the effect of fatty acids on ion channel associated proteins. To determine the mechanism of action of fatty acids on the activity of BK channels from rabbit pulmonary artery smooth muscle cells, all of the above mentioned mechanisms were considered. Most of the experiments described here were carried out using the patch-clamp technique and current recordings were performed in cell free, excised inside-out or outside-out membrane patches, in the absence of any added nucleotides and calcium. As a first step towards understanding how fatty acids modulate BK channel activity, as well as the type of protein site with which fatty acids may be interacting, we determined the structural features of the fatty acid molecule that are required for channel modulation. To do this the effects of a range of fatty acids and other lipids on BK channel activity were examined. The features required for BK channel activation were found to be the negatively charged head group and a carbon chain of greater than eight carbons. We also found that positively charged lipids produced the opposite effect of negatively charged lipids, a decrease in BK channel activity. A similar chain length requirement was also necessary for channel inhibition by positively charged lipids; short chain compounds did not alter activity while those with fourteen carbons or greater decreased activity. The identification of these required structural features suggested that a specific interaction between the charge on the lipid head group is required for channel modulation by these lipids. The requirement for a chain length of greater than eight carbons also suggests that a hydrophobic interaction is necessary for these lipids to be effective modulators of this channel. In addition, the identification of these required structural features makes it unlikely that modulation of BK channel activity by these lipid compounds is a consequence of a perturbation of the lipid environment in which the channel resides. Experiments were then carried out to determine whether modulation of BK channel activity by fatty acids and other charged lipids involved any of the following indirect mechanisms of action: 1) alterations in the concentration of calcium in the vicinity of the channel due to changes in membrane surface charge, or due to calcium stores attached to excised membrane patches, 2) alterations in the membrane electric field that the channel perceives due to changes in membrane surface charge and 3) changes in the activity of membrane bound protein kinases or protein phosphatases. In experiments where high ionic strength solutions were used to shield membrane surface charge, fatty acids and other charged lipids were still able to modulate BK channel activity suggesting that fatty acids do not act through a mechanism involving surface charge. Experiments carried out in high concentrations of EGTA (20 mM) make it unlikely that calcium is involved in the modulation of BK channels by fatty acids and other lipids. The involvement of membrane bound kinases or phosphatases is also unlikely as fatty acids effectively modulated BK channel activity in the presence of staurosporin, a kinase inhibitor, and okadaic acid, a phosphatase inhibitor. The elimination of these indirect and non-specific suggests that fatty acids and charged lipids modulate BK channel activity by directly interacting with, either the channel protein itself, or some other channel associated protein. To obtain further evidence that this indeed is the mechanism by which these lipids modulate BK channel activity; experiments were carried out to identify the site of action (i.e. side of the membrane) of both negatively and positively charged lipids. The negatively charged palmitoyl coenzyme A (PCoA) and a myristoylated positively charged peptide (myr-KPRPK), two compounds that are incapable of flipping across the bilayer, were used to identify the site of action of negatively and positively charged lipids. PCoA and myr-KPRPK produced their predicted effects of BK channel activation and suppression, respectively, only when they were applied to outside-out membrane patches. These experiments, therefore, support the contention that fatty acids and other charged lipids modulate BK channel activity by interacting with a site on the channel protein or a channel associated protein and that this site is found on the external membrane surface. If the site responsible for channel modulation by fatty acids and other charged lipids is contained within the BK channel protein itself, other members of this family may also possess this site, and thus be modulated by fatty acids. Experiments were performed, therefore, to determine whether the BK cloned channels, mslo, hslo and bslo could also be modulated by fatty acids. These cloned channels were expressed in the Xenopus oocytes, and whole-cell currents were recorded using the two-electrode voltage clamp technique. The fatty acids myristic and arachidonic acid were able to increase whole-cell current of oocytes expressing all clone types. The modulation of these cloned channels by fatty acids did not appear to involve calcium, the BK β-subunit or a bioactive metabolite of arachidonic acid. Although all possible mechanisms of action were not addressed in this study, the results support the idea that the site of fatty acid interaction resides in the channel protein itself. Taken together, therefore, these studies suggest that it is very likely that fatty acids and charged lipids modulate the activity of BK channels from smooth muscle cells of the rabbit pulmonary artery by directly interacting with an externally located site on the channel protein itself. The BK clones, mslo, hslo and bslo, are also modulated by fatty acids and it is likely that they share the same mechanism of action seen for BK channels from rabbit pulmonary artery smooth muscle cells.

Modulation of Ca2+ channels is an important mechanism for regulation of synaptic strength. However, it is clear that some Ca2+ current types are insensitive to inhibitory modulation mediated by heterotrimeric G proteins (G protein inhibition), and among currents which are sensitive to G protein inhibition, there is great variation in the magnitude of Ca2+ current inhibition between cells of different origin. For the experiments in this dissertation, I utilized recently cloned Ca2+ channels to determine the minimal combination of Ca2+ channel subunits which would confer G protein sensitivity to the recombinant channels. In addition, I examined the role Ca2+ channel auxiliary subunits play in regulation of Ca2+ channel sensitivity to inhibitory G proteins, and whether channels which were sensitive to G protein inhibition were regulated equivalently by the auxiliary subunits. Finally, I investigated possible mechanisms by which these auxiliary subunits modulate G protein-mediated inhibition of Ca2+ channels. I found that α1A and α1B Ca2+ currents, when expressed in Xenopus oocytes, were sensitive to modulation by G proteins in the absence of any Ca2+ channel auxiliary subunits, while α1C currents were not modulated under the same conditions. I conclude from this data that Ca2+ channel α1 subunits are differentially sensitive to G protein modulation, and the α1 subunit of the class A and B Ca2+ channels is sufficient for G protein modulation. I also tested the ability of Ca2+ channel auxiliary subunits to modulate the magnitude of G protein-mediated inhibition Ca2+ currents. I found that the Ca2+ channel α2 subunit had no effect on the magnitude of G protein inhibition of α1A and α1B currents. However, the Ca2+ channel β3 subunit eliminated tonic G protein inhibition and sharply reduced the magnitude of muscarinic M2 receptor induced G protein inhibition of both α1A and α1B currents. I found, however, that while the magnitude of α1A and α1B current inhibition was equivalent in the absence of auxiliary subunits, the magnitude of inhibition was greater for the α1B channel after co-expression of the Ca2+ channel β3 subunit. These results indicate that the Ca2+ channel β3 subunit reduces the sensitivity of α1A and α1B Ca2+ channels to voltage-dependent G protein modulation, and does so to a greater extent for α1A channels when compared to α1B Ca2+ channels. I found that M2 receptor induced inhibition of α1B currents was more voltage-dependent after expression of the Ca2+ channel β3 subunit. Additionally, the rate relief of G protein inhibition dramatically increased after co-expression of the Ca2+ channel β3 subunit. I also co-expressed G protein subunits, and determined that inhibition of both α1B and α1Bβ3 currents was mediated by the G protein βγ subunit. Furthermore, the rate of voltage-dependent relief of G protein βγ subunit induced inhibition increased after co-expression of the Ca2+ channel β3 subunit, similar to the increased rate of relief of the M2 receptor induced G protein inhibition. These data, along with data which demonstrates that G protein inhibition results from the binding of the G protein βγ subunit to the Ca2+ channel (De Waard et al., 1997), indicate that the Ca2+ channel β3 subunit subunit reduces the magnitude of G protein inhibition of α1B Ca2+ currents by increasing the rate of dissociation of the G protein βγ subunit, such that moderate depolarizations used to activate the channel also relieve a large portion of the G protein inhibition.

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

The current recommendations by the American Heart Association for health promotion are that all persons should partake in regular physical activity in order to reduce the risk of cardiovascular disease. Regular physical exercise reduces blood pressure and is an important component of the management of hypertension. It is therefore important that patients with hypertension participate in habitual physical exercise. Many hypertensive patients who exercise will require anti-hypertensive medication. However, some antihypertensive agents cause fatigue during exercise. In order for patients to gain the full benefits of an active lifestyle, it is important that the prescribed antihypertensive agent does not prevent them performing and enjoying sustained exercise. It has been well documented that β-blockers cause premature fatigue during physical exercise. The effects on exercise performance of other first line antihypertensive medications, such as calcium channel antagonists have not been extensively investigated. In particular, the effects of these agents on prolonged submaximal exercise endurance have not been well studied. The object of this thesis was to compare the effects of isradipine, a dihydropyridine calcium channel antagonist, to those of atenolol, a β₁-selective antagonist, on maximal and submaximal exercise performance and on short duration high-intensity exercise in physically active hypertensive patients. The study design was a crossover trial where drug treatments were double blinded and randomised. Physically active volunteers with mild to moderate hypertension were recruited. 11 subjects performed i) progressive exercise to exhaustion for determination of maximal oxygen consumption (VO₂max), maximal work load and cardiorespiratory responses to maximal exercise, ii) prolonged submaximal exercise for determination of exercise endurance, cardiorespiratory responses and ratings of perceived exertion (APE), and iii) short duration, high intensity exercise consisting of a 30 second maximal exercise test (Wingate test) to determine skeletal muscle power output, following 4 weeks ingestion of isradipine (2.5mg bd), atenolol (50mg bd) or placebo. Diastolic blood pressure at rest was reduced by both atenolol and isradipine, but was lowered to a greater extent by atenolol (83.3 vs 89.0 vs 96.1 mmHg, atenolol vs isradipine vs placebo, p<.0005). Systolic blood pressure at rest tended to be similarly reduced by both agents, but was significantly reduced during maximal and submaximal exercise by atenolol only (p<.001, atenolol vs isradipine, placebo). Heart rate at rest and during maximal and submaximal exercise was decreased by atenolol only (p<.0005, atenolol vs isradipine, placebo). Maximal exercise performance was reduced after atenolol ingestion compared to placebo but not after isradipine ingestion. Peak workload achieved during the maximal exercise test was decreased after atenolol but unchanged after isradipine ingestion (214 vs 243 W, atenolol vs placebo, p<.01). Similarly, VO₂max was reduced after atenolol compared to placebo but was unchanged after isradipine ingestion (33.6 vs 36.4, 33.6 vs 36.1 mlO₂/kg/min, atenolol vs placebo, atenolol vs isradipine, p<.05). Both atenolol and isradipine ingestion reduced submaximal endurance time compared to placebo (27.8 vs 46.4, 34.4 vs 46.4 min, atenolol vs placebo, isradipine vs placebo, p<.005), and increased rating of perceived exertion (APE) after 30 min of submaximal exercise (p<.05). Submaximal oxygen consumption (VO₂), ventilation, respiratory exchange ratio (REA) and blood lactate, glucose and free fatty acid concentrations were not altered after the ingestion of either agent. Neither agent influenced peak skeletal muscle power, total work done, or rate of fatigue during the Wingate test compared to placebo. The results of these studies indicate that impaired performance and increased RPE during submaximal exercise after ingestion of either atenolol or isradipine is not due to alterations of ventilation, VO₂, RER, or blood lactate, glucose and free fatty acid concentrations during prolonged submaximal exercise. Similarly, reduced submaximal exercise performance after atenolol or isradipine ingestion is not due to factors which would also limit the ability of skeletal muscle to perform short duration, high intensity exercise before a bout of prolonged exercise. This study demonstrates that prolonged submaximal exercise testing can reveal an impairment in exercise performance after ingestion of antihypertensive medication which is not evident during maximal exercise testing. This finding is important as prolonged submaximal exercise is the form of exercise which most hypertensive patients actually perform. Further research is required on the effects of anti-hypertensive medications on submaximal exercise performance before firm recommendations can be made regarding medications most suitable for the physically active hypertensive patient. The results of these and other studies indicate that it is not yet possible to make claims that the calcium channel antagonist agents are without effect on physical exercise performance in physically active hypertensive patients.