Dopamine transporters (DAT) facilitate high-affinity presynaptic dopamine (DA) reuptake in the central nervous system, and are required to constrain extracellular DA levels and maintain presynaptic DAergic tone. DAT is the primary target for addictive and therapeutic psychostimulants, which require DAT binding to elicit reward. DAT availability at presynaptic terminals ensures its proper function, and is dynamically regulated by endocytic trafficking. My thesis research focused on two fundamental questions: 1) what are the molecular mechanisms that control DAT endocytosis? and 2) what are the mechanism(s) that govern DAT’s post-endocytic fate? Using pharmacological and genetic approaches, I discovered that a non-receptor tyrosine kinase, activated by cdc42 kinase 1 (Ack1), stabilizes DAT plasma membrane expression by negatively regulating DAT endocytosis. I found that stimulated DAT endocytosis absolutely requires Ack1 inactivation. Moreover, I was able to restore normal DAT endocytosis to a trafficking dysregulated DAT coding variant identified in an Attention Deficit Hyperactivity Disorder (ADHD) patient via constitutively activating Ack1. To address what mechanisms govern DAT’s post-endocytic fate, I took advantage of a small molecule labeling approach to directly couple fluorophore to the DAT surface population, and subsequently tracked DAT’s temporal-spatial post-endocytic itinerary in immortalized mesencephalic cells. Using this approach, I discovered that the retromer complex mediates DAT recycling and is required to maintain DAT surface levels via a DAT C-terminal PDZ-binding motif. Taken together, these findings shed considerable new light on DAT trafficking mechanisms, and pave the way for future studies examining the role of regulated DAT trafficking in neuropsychiatric disorders.

Proton fluxes through plasma membranes are essential for regulating intracellular and extracellular pH and mediating co-transport of metabolites and ions. Although conventional electrical measurements are highly sensitive and precise for proton current detection, they provide limited specificity and spatial information. My thesis focuses on developing optical approaches to visualize proton fluxes from ion channels and transporters. It has been demonstrated that channel-mediated acid extrusion causes proton depletion at the inner surface of the plasma membrane. Yet, proton dynamics at the extracellular microenvironment are still unclear. In Chapter II, we developed an optical approach to directly measure pH change in this nanodomain by covalently attaching small-molecule, fluorescent proton sensors to the cell’s glycocalyx using glyco-engineering and copper free ‘click’ chemistry. The extracellularly facing sensors enable real-time detection of proton accumulation and depletion at the plasma membrane, providing an indirect readout of channel and transporter activity that correlated with whole-cell proton current. Moreover, the proton wavefront emanating from one cell was readily visible as it crossed over nearby cells. The transport of monocarboxylates, such as lactate and pyruvate is critical for energy metabolism and is mainly mediated by proton-coupled monocarboxylate transporters (MCT1-MCT4). Although pH electrodes and intracellular fluorescent pH sensors have been widely used for measuring the transport of proton-coupled MCTs, they are unable to monitor the subcellular activities and may underestimate the transport rate due to cell’s volume and intracellular buffering. In Chapter III, we used the Chapter II approach to visualize proton-coupled transport by MCT1-transfected HEK293T cells and observed proton depletion followed by a recovery upon extracellular perfusion of L-lactate or pyruvate. In addition, we identified a putative MCT, CG11665/Hrm that is essential for autophagy during cell death in Drosophila. The results demonstrate that Hrm is a bona fide proton-coupled monocarboxylate transporter that transports pyruvate faster than lactate. Although the approach developed in Chapter II enables visualization of proton fluxes from ion channels and transporters, it’s not applicable in some cell types which cannot incorporate unnatural sialic acid precursors into their glycocalyx, such as INS-1 cells and cardiomyocytes. To address this, in Chapter IV we developed a pH-sensitive, fluorescent WGA conjugate, WGA-pHRho that binds to endogenous glycocalyx. Compared to the results in Chapter II and III, cell surface-attached WGA-pHRho has similar fluorescent signals in response to proton fluxes from proton channel Hv1, omega mutant Shaker-IR R362H and MCT1. With WGA-pHRho, we were able to label the plasma membrane of INS-cells and cardiomyocytes and visualized the transport activity of MCT1 in these cells. Taken together, these findings provide news insights into proton dynamics at the extracellular environment and provide new optical tools to visualize proton fluxes from ion channels and transporters. Moreover, the modularity of the approaches makes them adaptable to study any transport events at the plasma membrane in cells, tissues, and organisms.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Human BCRP is one of the major ATP-binding cassette transporters involved in the development of multidrug resistance in cancer chemotherapy. Overexpression of BCRP in the tumor cell plasma membrane and apical membrane of the gastrointestinal tract leads to decreased intracellular accumulation of various anticancer drugs as well as reduced drug bioavailability. BCRP has been shown to exist on the plasma membrane as higher forms of homo-oligomers. In addition, the oligomerization domain of BCRP has been mapped to the carboxyl-terminal TM5-ECL3-TM6 and this truncated domain, when co-expressed with the full-length BCRP, displays a dominant inhibitory activity on BCRP function. Thus, the oligomerization of BCRP could be a promising target in reversing multidrug resistance mediated by BCRP. To further dissect the oligomerization domains of human BCRP and test the hypothesis that TM5, ECL3, and TM6 each plays a role in BCRP oligomerization and function, we engineered a series of BCRP domain-swapping constructs with alterations at TM5-ECL3-TM6 and further generated HEK293 cells stably expressing wild-type or each domain-swapping construct of BCRP. Using co-immunoprecipitation and chemical cross-linking, we found that TM5, ECL3, and TM6 all appear to partially contribute to BCRP oligomerization, which are responsible for the formation of oligomeric BCRP. However, only TM5 appears to be a major contributor to the transport activity and drug resistance mediated by BCRP, while ECL3 or TM6 is insufficient for BCRP functions. Taken together, these findings suggest that homo-oligomeric human BCRP may be formed by the interactions among TM5, ECL3 and TM6, and TM5 is a crucial domain for BCRP functions and BCRP-mediated drug resistance. These findings may further be used to explore targets for therapeutic development to reverse BCRP-mediated drug resistance and increase the bioavailability of anti-cancer drugs for better treatment of multidrug resistant cancers.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Júlia Jurčáková Supervisor: RNDr. Jakub Hofman PhD. Title of diploma thesis: The assessment of inhibitory effects of selected targeted anticancer drugs on the activity of ABC drug eflux trasporters. Lung cancer is the leading cause of death within oncological diseases. Non-small cell lung carcinoma (NSCLC) accounts for about 85% of all lung cancer, and its major subtypes include adenocarcinoma and squamous cell carcinoma. In addition to surgery, radiotherapy and chemotherapy, the use of targeted low-molecular substances, which target tumor cells with higher specificity, has recently been used in treatment. The two main causes of death in cancer patients are the formation of metastases and the development of multidrug resistance (MDR). This may also be caused by overexpression of the efflux transporters. ATP-binding cassette (ABC) transporters are groups of transmembrane pumps that use energy in the form of ATP to transfer a wide range of substrates. In particular, P-glycoprotein (ABCB1), breast cancer-resistance protein (ABCG2) and multidrug resistance-associated protein 1 (ABCC1) are associated with MDR. Inhibition of these transporters increases the amount of cytostatic substrate within the...

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0-60min after pre-treatment with α-CCA-Na in mice (-47%) and in piglets (-66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

No description available.

Les pyréthrinoïdes et les organophosphorés sont des pesticides très utilisés, à l’origine d’une imprégnation forte de la population, exposée à ces contaminants principalement via l’alimentation. De plus en plus d’études scientifiques suggèrent des liens entre l’exposition à ces composés et des maladies chroniques ou des troubles du développement de l’enfant. Paradoxalement, leur devenir biologique chez l’homme est mal connu. Certaines études suggèrent que ces insecticides sont susceptibles d’intéragir avec les transporteurs membranaires ABC et SLC, protéines localisées au niveau d’interfaces hémato-tissulaires qui prennent en charge de nombreux substrats endogènes, médicaments et contaminants de l’environnement. L’objectif de notre étude a été de caractériser les effets d’insecticides des familles des pyréthrinoïdes et des organophosphorés sur l’activité de nombreux transporteurs ABC et SLC prenant en charge des médicaments (P-gp, BCRP, MRPs, OATP-1B1,-2B1,-1B3, OCT1-3, OAT1, OAT3, MATE1 et MATE2K) par une approche in vitro. Nous nous sommes également attachés à caractériser par des expérimentations in vitro et in silico, les mécanismes des interactions et les éléments structuraux des pesticides à l’origine de ces effets. Nous avons montré que de nombreux organophosphorés et pyréthrinoïdes étaient capables d’inhiber des transporteurs d’efflux (MRP, BCRP, P-gp) et d’influx (OATP1B1, OAT3, MATE1, OCT1-2) et de stimuler l’activité de certains OATPs. Les pesticides testés inhibaient très fortement l’activité des transporteurs de cations (OCT1 et OCT2) et ont pu bloquer le transport de catécholamines médiés par ces protéines. Une approche qSAR a permis de définir des paramètres physicochimiques associés aux effets modulateurs des pesticides et une approche d’amarrage moléculaire (docking) a mise en évidence les sites de liaisons de la P-gp impliquées dans ces interactions. Les conséquences des modulations de l’activité des transporteurs, en termes d’effets toxiques et d’interactions médicamenteuses, restent à définir pour les populations exposées à de fortes doses de pesticides. Toutefois, la contribution des interactions observées aux effets toxiques de ces insecticides est peu probable car nécessitant des concentrations nettement supérieures à celles atteintes dans le cadre d’une exposition environnementale de la population générale. / The general population is chronically exposed to pyrethroids and organophosphorus insecticides, mainly through alimentation. Several epidemiological studies have found an association between non-occupational exposure to these pesticides and chronic diseases and developmental disorders. Paradoxically, their biological fate in humans is poorly understood. Some studies suggest that these insecticides could interact with ABC and SLC membrane transporters. These membrane proteins, located at blood-tissue interfaces (liver, kidney, intestine ...), handle many endogenous substrates, drugs and pollutants. The objective of our study was to characterize, using an in vitro approach, the effects of pyrethroid and organophosphorus insecticides on the activity of numerous ABC and SLC human drug-transporters (P-gp, BCRP, MRPs, OATP-1B1, -2B1, -1B3, OCT1-3, OAT1, OAT3, MATE1 and MATE2K). We have also tried to analyze the mechanisms of interactions and the structural requirements for insecticides-mediated modulation of drug transporters activities using in vitro and in silico approach. We have shown that many organophosphorus and pyrethroids are able to inhibit ABC (MRP, BCRP, P-gp) and SLC (OATP1B1, OAT3, MATE1, OCT1-2) transporters and can stimulate the activity of some OATPs. Moreover, the tested pesticides inhibited very strongly the activity of OCT1 and OCT2 and blocked catecholamine transport mediated by these transporters. A qSAR approach allowed to define physicochemical parameters associated with the modulating effects of pesticides and a molecular docking approach revealed the P-gp binding sites involved in these interactions. The consequences of transporter activitie modulation, in terms of toxic effects and drug interactions, remain to be defined for populations exposed to high doses of pesticides, occurring notably in response to poisoning. However the alterations of these transporter activities by insecticides are unlikely to contribute to organophosphorus or pyrethroids toxicities of chronic low-dose exposure.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ronette Gehring / Milk composition has a dynamic nature, and the composition varies with stage of lactation, age, breed, nutrition and health status of the udder. The changes in milk composition seem to match the changes in the expression of membrane proteins in secretory mammary epithelial cells that are needed for the movement of molecules from blood to milk and vice versa (Nouws and Ziv, 1982). Thus, an understanding of transporter expression, function and regulation in mammary epithelial cells can provide insight into mammary gland function and regulation. The goal of this project was to elucidate (molecularly and functionally) the role of drug transporters in the barrier function of an epithelial monolayer cultured from an immortalized bovine mammary epithelial cell line (BME-UV). To characterize the regulation (expression and function) of these drug transporters in BME-UV cells after exposure to cytokine TNF-α for selected periods of time. Representative members of drug transporters of the SLC (OCT and OAT) and ABC (P-glycoprotein) superfamilies were chosen for this project. In the first study, the involvement of a carrier-mediated transport system in the passage of organic cation (TEA) and anion (EsS) compounds was elucidated across the BME-UV monolayer. In the second study, molecular and functional expression of bOAT isoforms in BME-UV cells were studied. The final study characterized the effects of cytokine TNF-α on the expression and function of P-glycoprotein, an efflux pump, in BME-UV cells. Cytokine TNF-α exposure induced the expression of ABCB1 mRNA and increased P-glycoprotein production in BME-UV cells, resulting in a greater efflux of digoxin, a known P-glycoprotein substrate, back into the apical fluid. The expression, function, and regulation of these transporters in the mammary gland has important implications for understanding the barrier function of the mammary epithelium and, in more specific, for characterizing the role of these transporters in the accumulation and/or removal of specific substrates from milk and/or plasma. Moreover, this study provides an in vitro cell culture model of mammary epithelium to characterize mammary epithelial cell function during inflammation.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical (HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter protein function and contribute to the onset of myocardial insulin resistance and impaired contractile function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation, and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4 translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose. Although our results were not conclusive, the data suggest a potential novel link between dexamethasone exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and related metabolic derangements and diseases), these results provide new targets for potential therapeutic agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier (HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie. Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak, die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê. Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4) translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4 translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie. Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.