T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs. / Medicine, Faculty of / Graduate

The purpose of my study was to determine and compare the effects of three calcium antagonists on systemic hemodynamics, ECG, blood flow distribution, tissue conductance and venous tone of the rat. The effects of a representative drug from Spedding's (1985) three subclasses of calcium antagonists on systemic hemodynamics, ECG, cardiac output and the distribution of blood flow were investigated by the microsphere technique in pentobarbital-anesthetized rats. The representative drugs were: I, nifedipine (12 and 35 µg/kg/min); II, verapamil (43 and 83 µg/kg/min) and III, flunarizine (174 and 275 µg/kg/min). Low and high doses were selected to give a decrease in mean arterial pressure of 10 and 20 mmHg, respectively, compared with control rats. At equal depressor levels, all the drugs similarly decreased total peripheral resistance while slightly but not significantly increasing cardiac output (CO) and stroke volume. Heart rate was decreased by verapamil and flunarizine, but increased by nifedipine. The high dose of nifedipine decreased contractility as measured by dP/dt and had no effect on PR-interval, while verapamil decreased dP/dt and prolonged the PR-interval. The low dose of nifedipine and both doses of flunarizine slightly but not significantly decreased dP/dt and had no effect on PR-interval. All three drugs similarly affected the distribution of blood flow. Blood flow to lungs, liver, and heart was increased while flow to the intestine, kidneys, spleen and skin was decreased. Arterial conductances in lungs, liver, heart and skeletal muscle were increased by the three drugs. These results show that representative drugs from the three subclasses of calcium antagonists had similar effects on the distribution of blood flow and arterial conductances but different chronotropic, dromotropic and inotropic effects. A final set of experiments were designed to evaluate calcium antagonist actions on venous tone, as venous tone is a primary determinant of CO and the calcium antagonists generally increase CO. The effects of three calcium antagonists, verapamil, nifedipine and flunarizine on mean arterial pressure (MAP), heart rate (HR) and mean circulatory filling pressure (MCFP), an index of total body venous tone, were investigated in the. conscious rat. Infusions of all three drugs caused a dose-dependent decrease in MAP and an increase in MCFP, compared with the corresponding values in control rats. HR was decreased by verapamil and flunarizine and slightly increased by nifedipine. Further experiments investigated whether the increase in MCFP by verapamil was indirectly caused by reflex activation of the autonomic nervous system. Rats were pretreated with a continuous infusion of the ganglionic blocker hexamethonium prior to infusion of verapamil. After treatment with hexamethonium, verapamil did not increase the MCFP. In fact the highest dose of verapamil significantly decreased MCFP. The results suggest that calcium antagonists have greater dilator effects in arterioles compared to veins. It appears that any direct venodilator effects of verapamil in conscious rats are masked due to reflex activation of the autonomic nervous system. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Le récepteur du facteur de croissance épidermique (EGFR) est une cible rationnelle pour les traitements anticancéreux des carcinomes épidermoïdes (CE). Cependant, seule une petite fraction de patients présente des avantages cliniques. Afin de comprendre l’échec de ces thérapies, j’ai étudié la voie de signalisation de l’EGFR en présence de fibroblastes associés aux carcinomes (FAC), principales cellules non malignes au sein des tumeurs. J'ai démontré que dans les CE, la voie de signalisation de l’EGFR coopère avec la rigidité de la matrice extracellulaire (MEC) induite par les FAC. Par la suite, j'ai cherché à résoudre les voies moléculaires qui sous-tendent cette coopération afin d'identifier de nouvelles cibles pharmaceutiques. Grâce à un criblage d'inhibiteurs pharmacologiques, j’ai identifié le vérapamil et le diltiazem, bloqueurs des canaux calcique CaV1, comme étant de puissants inhibiteurs de l'invasion des CE. Au niveau moléculaire, j'ai révélé que la rigidité de la MEC dérivée de la tumeur et la signalisation de l'EGFR déclenchent l'augmentation du calcium intracellulaire par le canal CaV1.1 dans les CE. Le blocage de l'activité de ces canaux inhibe l’invasion et la prolifération des cellules tumorale in vitro. Plus important encore, je démontre une forte réduction du développement des tumeurs dans deux modèles in vivo, à la fois dans un modèle de xénogreffe de cellules dérivées de patient atteint de carcinome de la tête et du cou, et dans un modèle CE cutané chez la souris. Par conséquent, je suggère une réaffectation du vérapamil et du diltiazem en tant qu’agents anticancéreux. / Epidermal growth factor receptor (EGFR) is a rational target for squamous cell carcinoma (SCC) anticancer therapies, nevertheless; only a subset of patients shows clinical benefits. I demonstrated a cooperation between EGFR signaling and extracellular matrix (ECM) stiffness that could explain this phenomenon. I sought to resolve the molecular pathway underlying this cooperation in SCC proliferation and expansion in order to identify new pharmaceutical targets. Screening of pharmacological inhibitors, in an in vitro 3-D assay, identified verapamil and diltiazem, FDA approved L-type calcium channels inhibitors, as potent blockers of SCC invasion. Mechanistically, I revealed that tumor-derived ECM stiffness and EGFR signaling trigger increased of intracellular calcium through the L-type CaV1.1 channel in SCC. Blocking L-type calcium channels activity resulted in reduced SCC cells invasion and proliferation in vitro. More importantly, I also demonstrate a strong reduction in tumor development in two in vivo models, both head and neck patient derived xenograft and skin SCC mice model. Consequently, I suggest a repurpose of verapamil and diltiazem to anti-cancer agents.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

<p>  </p> <p>Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood. </p> <p>To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that <em>INS2</em> transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells</p> <p>We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.</p> <p>Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.</p>

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Cardiovascular disease remains the number one cause or mortally in the western world. Heart failure is the most rapidly growing cardiovascular disease (Hobbs, 2004; Levy, et al., 2002). Heart failure, by definition, is progressive deteriorating function of the heart due to progressive cardiac myocytes loss. Though after decades of endeavor of searching the pathophysiology and treatments for heart failure, it remains highly lethal. Therefore, it is vital to find novel therapies to help treat such chronic disease. Replace the lost cardiomyocyte with new ones could restore cardiac function and reduce mortality. The purpose of this study is to investigate on how TTCCs (T-type calcium channels) affect cardiomyocyte proliferation. In mice after birth, the major TTCC expressed in the heart is Cav3.1/α1G, and therefore we used Cav3.1/α1G transgenic (TG), knockout (-/-) and wild type mice respectively to define the role of TTCC in cardiomyocyte proliferation. In neonatal mouse ventricular myocyte (NMVMs) right after birth, there is almost no TTCC after birth in α1G-/- NMVMs, whereas there are around 35% NMVMs in wild type (WT) show TTCC. On day 7 after birth, there are no T-type calcium currents in both α1G-/- NMVMs and WT NMVMs. Using BrdU, a DNA synthesis marker, we identified plenty of BrdU positive cardiomyocyte during the first seven days after birth. Cardiomyocyte is special due to its double nucleation property. Our cell cycle studies showed that there is significant difference in cell cycle distribution between α1G-/- and WT NMVMs on day seven after birth. Significantly more NMVMs are arrested in G1 phase in α1G-/-, compared to WT NMVMs. Even until 2 month old, there are still significantly more mono-nucleated cardiomyocyte in α1G-/- than in WT. In conclusion, all these evidence showed that blocking T-type calcium channel could partially prevent binucleation from happening and stop cardiomyocytes withdrawal from cell cycle. Mononucleated cardiomyocyte is still able to proliferate. Hence, mononucleated cardiomyocytes in adult still have potential to proliferation because these cardiomyoctes are arrested in their cell-cycle before their terminal differentiation, which could offer a novel approach for cardiac repair. / Physiology

No / T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S. / This work was supported by the British Heart Foundation (PG/11/84/29146).