596 research outputs found

    The Effect of Oxytocin Receptor Genetic Variants on Oxytocin Response

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    Oxytocin is a nonapeptide hormone that modulates social behavior, mediates the lactation reflex, and induces and strengthens uterine contractions. Each year, oxytocin is administered to about half of laboring patients for induction and augmentation, and to almost all patients for prevention of post-partum hemorrhage – a total of four million patients per year in the United States alone. However, response to oxytocin varies widely between individuals, and complications including uterine hyperstimulation, uterine atony, required Cesarean section, and postpartum hemorrhage can arise from either excessive or inadequate oxytocin response. To avoid these adverse events, it is critically important to identify individuals at risk for aberrant oxytocin response and develop strategies to improve outcomes in these patients.In this dissertation, I first sought to determine the effects of common missense genetic variants in the oxytocin receptor (OXTR) gene on oxytocin response. I screened prevalent genetic variants in OXTR and found that five variants alter calcium signaling and β-arrestin recruitment in cells. Three of these five variants had differential effects on calcium signaling and β-arrestin recruitment, leading to imbalance between activation and desensitization of the OXTR. Molecular dynamics simulations showed that these three variants cause conformational changes that are consistent with the signaling changes we observed in cells. Overall, these data show that OXTR variants alter oxytocin response, and suggest that individuals with these genetic variants may benefit from personalized oxytocin dosing. In the second part of this dissertation, I investigated the role of OXTR trafficking in determining oxytocin responsiveness. OXTR genetic variants that impair OXTR trafficking decrease maximal oxytocin response in transfected cells. I investigated whether small molecule modulators of OXTR and the closely related vasopressin receptor family could act as pharmacological chaperones for OXTR, increasing the cell surface localization of the wild type OXTR and rescuing the trafficking defects in variant OXTR. Treatment with the oxytocin/vasopressin antagonists increased cell surface localization of variant OXTR and rescued oxytocin signaling. The increase in OXTR cell surface localization and oxytocin responsiveness could also be observed in immortalized and primary human myometrial cells. Overall, these results show that pharmacological chaperones can enhance trafficking and function for both wild type and variant OXTR. This work presents a novel therapeutic strategy to improve the efficacy of oxytocin use in patients predicted to respond poorly due to genetic or environmental factors

    New characterization of dihydroergotamine receptor pharmacology in the context of migraine: utilization of a β-arrestin recruitment assay

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    IntroductionDihydroergotamine mesylate (DHE) is an established effective acute therapy for migraine and is often characterized by its broad receptor pharmacology. Knowledge of DHE pharmacology largely comes from studies employing older methodologies.ObjectiveTo assess DHE receptor activity using high-throughput methods to screen for functional ß-arrestin activity at G protein–coupled receptors (GPCRs).MethodsFunctional receptor activities of DHE and sumatriptan succinate (both 10 μM) were screened against 168 GPCRs using the gpcrMAX assay. Agonist and antagonist effects were considered significant if receptor activity was >30% or inhibited by >50%, respectively. Radiolabeled ligand binding assays were performed for DHE (0.01–300 nM for 5-HT3 and 4E; 0.3–10,000 nM for 5-HT1B, α-adrenergic2B [i.e., α2B-adrenoceptor], D2, and D5) to assess specific binding to select receptors.ResultsDHE (10 μM) exhibited agonist activity at α-adrenergic2B, CXC chemokine receptor 7 (CXCR7), dopamine (D)2/5, and 5-hydroxytryptamine (5-HT)1A/1B/2A/2C/5A receptors and antagonist activity at α-adrenergic1B/2A/2C (i.e., α1B/2A/2C-adrenoceptors), calcitonin receptor–receptor activity modifying protein 2 (CTR-RAMP2) or amylin 2 (AMY2), D1/3/4/5, and 5-HT1F receptors. Sumatriptan succinate (10 μM) exhibited agonist activity at the 5-HT1B/1E/1F/5A receptors. DHE demonstrated a half-maximal inhibitory concentration (IC50) of 149 nM at the 5-HT1F receptor and a half-maximal effective concentration (EC50) of 6 μM at the CXCR7 receptor. DHE did not bind to the 5-HT3 receptor at concentrations up to 300 nM and bound poorly to 5-HT4E and D5 receptors (IC50 of 230 and 370 nM, respectively). DHE bound strongly to the D2, 5-HT1B, and α-adrenergic2B receptors (IC50 of 0.47, 0.58, and 2.8 nM, respectively).ConclusionBy using a high-throughput β-arrestin recruitment assay, this study confirmed the broad receptor profile of DHE and provided an update on DHE receptor pharmacology as it relates to migraine

    Novel pharmacological tools for investigating the pharmacology of free fatty acid receptor 2

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    The G protein-coupled receptor FFA2 acts as a receptor for short chain fatty acids produced by commensal bacteria through the fermentation of dietary fibre. While FFA2 is implicated in several disease conditions, such as obesity and ulcerative colitis, the receptor has so far not been successfully exploited therapeutically. This may reflect marked species selectivity of antagonists for the human receptor, which hinder pre-clinical in vivo studies in rodent models of disease. Herein a novel transgenic mouse strain expressing human FFA2 with an integrated HA epitope tag (hFFA2-HA) was characterised as a novel animal model. To assess the expression of hFFA2-HA at the messenger RNA and protein levels, quantitative reverse-transcription PCR and immunohistochemistry approaches were utilised. hFFA2-HA was found to be expressed in adipose, colon, spleen and bone marrow-derived neutrophils, at equivalent levels as mouse FFA2 in C57BL/6N mice. In addition, hFFA2-HA expression was confirmed in subsets of enteroendocrine and haematopoietic cells in the intestine and spleen, respectively. Following immunocytochemical confirmation of hFFA2-HA protein expression in bone marrow-derived neutrophils, these were used in functional studies ex vivo. The functionality of hFFA2-HA on neutrophils was established with the use of the hFFA2-selective radioligand [3H]GLPG0974, which was found to specifically bind membranes from hFFA2-HA mouse neutrophils, and with the use of the endogenous ligand propionate in [35S]GTPγS incorporation assays, where a concentration-dependent response was observed. Importantly, hFFA2-selective antagonists were able to inhibit C3-induced [35S]GTPγS incorporation and chemotaxis in hFFA2-HA-expressing, but not in C57BL/6N neutrophils. For the investigation of hFFA2 phosphorylation, novel, potentially phosphosite-specific antisera were evaluated through Western Blot and immunocytochemistry methods. Here, residues Thr306 and Thr310 were observed to be phosphorylated in an agonist-dependent manner, while residues Ser296 and Ser297 appeared to be constitutively phosphorylated. Replacement of these residues with alanine abolished antiserum binding but did not fully ablate β-arrestin-2 interactions. The development of novel pharmacological research tools, here in the form of transgenic mouse models and phosphosite-specific antisera, can not only aid understanding of FFA2 signalling and pharmacology but can also bridge the translational gap between in vitro and clinical studies

    Ligand and G-protein selectivity in the Îş-opioid receptor

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    The Îş-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorder

    Effect of TBBPA on arterial contractile regulation and possible implications for the development of hypertensive diseases

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    The endocrine disruptor (EDCs) is a compound that has been defined as “an exogenous agent that interferes with the production, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes.” This compound can affect the endocrine function via interference with hormone pathways (e.g., oestrogen, androgen, or thyroid hormone). The constant human exposure to endocrine disruptors has raised some concerns. Some of these components are suspected of being harmful to human health. Brominated flame retardants (BFRs) are chemicals widely used in consumer products, including electronics, vehicles, plastics, and textiles, to reduce flammability. These compounds can interfere with hormone homeostasis, so they are considered endocrine disruptors. Tetrabromobisphenol A (TBBPA) is the most studied BFRs due to its toxicity and presence in a variety of environmental media and the human being. The exposure to this compound is associated with several health risks: thyroid disorders, diabetes, reproductive health, cancer, and neurobehavioral development disorders. In addition, TBBPA exposure can be correlated with some cardiovascular disorders, such as diabetes and obesity. This compound has also been detected in biological samples such as human serum, urine, and breast milk. Moreover, TBBPA has also been detected in the umbilical cord of Japanese pregnant women, proving a prenatal exposure to this compound. This observation suggests that TBBPA can cross the human placenta. In this scenario, it is important to understand how the TBBPA exposure effects the vascular tonus and if the endocrine disrupting effects from that exposure can be detected in future generations. In this project, organ bath and patch clamp techniques were developed and applied to achieve the main goal of this doctoral thesis: to study the effect of TBBPA on arterial contractility and analyse the mode of action of TBBPA as a human EDCs and understand its involvement in vascular disorders. This study was performed in two different study models: in the human umbilical artery (HUA) and in the rat aorta. Additionally, the cGMP compartmentation in human vascular smooth muscle was also analysed. Therefore, in the first research work presented, we infected smooth muscle cells with adenovirus containing mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel-subunit to understand how the cGMP conveys different information and we recorded the associated cGMP-gated current (ICNG). The whole cell configuration of the patch clamp technique was used to measure the ICNG and the potassium current (IK) in human umbilical artery smooth muscle cells (HUASMC). Atrial Natriuretic Peptide (ANP) induced an activation of basal ICNG, whereas sodium nitroprusside (SNP) had a slight effect. IBMX (nonselective PDE inhibitor), T0-156 (PDE5 inhibitor), and cilostamide (PDE3 inhibitor) all had a small effect on the basal ICNG current. Concerning potassium channels, we observed that ANP and testosterone induced activation of IK and this effect is bigger than that induced by SNP, cilostamide and T0-156. Cilostamide and T0-156 decreased the ICNG stimulation induced by ANP and testosterone, suggesting that the pGC pool is controlled by PDE3 and PDE5. Thus, the effects of SNP show the presence of two separated pools, one next to the plasma membrane and controlled by the PDE5 and PDE3, and a second pool in the cytosol of the cells that is regulated mainly by PDE3. These findings show the existence of cGMP compartmentalization in human vascular smooth muscle cells, and this phenomenon is controlled by PDE3 and PDE5. The second research work evaluated the direct effects and the 24 h exposure of TBBPA on the HUA and also its mode of action (MOA). The viability of HUASMC was analysed using MTT assay and the cells exposed to high concentrations of TBBPA (500 and 1000 μM) showed a decrease in cell viability. Using the organ bath technique, endothelium-denuded HUA rings were contracted with serotonin (5-HT), histamine (His), and potassium chloride (KCl), and then the direct effects of TBBPA (0.01- 100 μM) were analysed. The effects of 24 hours TBBPA exposure (1, 10, and 50 μM) were also analysed on contractile responses of HUA to 5-HT, His, and KCl. Furthermore, the vascular MOA of TBBPA was studied through the analysis of cGMP and calcium (Ca2+) channels activity, these pathways are involved in the relaxation and contraction of HUA, respectively. Our results demonstrated that the direct effects of TBBPA induce a vasorelaxation of HUA. The 24h TBBPA exposure changed the vasoconstrictor response pattern of 5-HT, His and KCl and the vasorelaxant response pattern of SNP and nifedipine. This effect is due to the involvement of TBBPA with the NO/sGC/cGMP/PKG pathway and the interference in Ca2+ influx. Furthermore, using the real-time quantitative polymerase chain reaction (RT-qPCR), TBBPA clearly modulates L-type Ca2+ and large-conductance Ca2+ 1.1 α- and β1 subunit channels, and soluble guanylyl cyclase (sGC) and protein Kinase G. In this sense, our data demonstrated that TBBPA induces changes in the vascular homeostasis of HUA. In the last part of this work, the effect of TBBPA in rat aortic smooth muscle and the possible mechanisms involved were investigated and to achieve these goals, we started with the analysis of A7r5 cells viability. These cells were exposed to different TBBPA concentrations, and the results showed that the high concentrations of TBBPA (500 and 1000 μM) decreased the viability of the A7r5 cells. Then, using the organ bath technique, rat aorta rings without endothelium were contracted with Phenylephrine, Noradrenaline, and isosmotic KCl solution to evaluate the vascular effect of TBBPA (0.01–100 μM). Furthermore, MOA of TBBPA was studied through Nifedipine (specific blocker of L-type VGCC), tetraethylammonium (TEA), 4-aminopyridine (4-AP), and glybenclamide (Gly) (K+ channel inhibitors). Our results suggest that the direct effects of TBBPA induced vasorelaxation of rat aorta, involving the inhibition of Ca2+ channels and activation of potassium channels. Moreover, through RT-qPCR, it was demonstrated that TBBPA clearly modulates L-type Ca2+ and large-conductance Ca2+ 1.1 α- and β1 subunit channels, and sGC and protein Kinase G. Overall, it was shown that TBBPA exposure also interferes with vascular homeostasis of rat aorta through Ca2+ and K+ channels. In conclusion, the main findings of this thesis confirmed the crucial actions of TBBPA in vascular smooth muscle. These effects demonstrate that TBBPA induces smooth muscle relaxation through an endothelium-independent MOA. Due to sGC activation that increases the cGMP intracellular levels, inhibition of L-Type VGCC and activation of K+ channels were verified. Another innovative result of the present thesis was the identification of cGMP compartmentalization in human vascular smooth muscle cells. Further understanding and targeting of these results might be exploited in future studies to acknowledge the effects of TBBPA at the vascular level and its complexity in environmental and human exposure.Disruptores endócrinos (EDCs) são substâncias exógenas ao corpo humano que podem interferir na síntese, secreção, transporte, metabolismo ou eliminação das diferentes hormonas, que são responsáveis pela manutenção da homeostase corporal, reprodução, desenvolvimento ou comportamento. Os EDC são um grupo muito heterogéneo de compostos, que vão desde químicos sintéticos a alguns produtos constituintes naturais de algumas plantas. A avaliação do seu impacto na saúde é extremamente difícil, assim a constante exposição humana a EDCs tem suscitado algumas preocupações. Sabe-se atualmente que existem diversas patologias em que estas substâncias podem ter um papel determinante como causadoras ou amplificadoras das suas manifestações, uma vez que esses compostos afetam a função endócrina interferindo nas vias hormonais (por exemplo: estrogénio, androgénio ou hormonas tiroidianas). Os retardadores de chama bromados (BFRs) são produtos químicos omnipresentes usados amplamente pela indústria. Estes compostos são frequentemente usados em eletrónica, veículos motorizados, brinquedos, plásticos e têxteis para reduzir a inflamabilidade. Os BFRs são lipofílicos e persistentes, e infelizmente muitas destas substâncias químicas não permanecem fixas no produto que as contém, sendo lentamente libertadas para o ar, para as partículas de pó e água, e terminam entrando nos alimentos e no nosso organismo. Os efeitos nocivos para a saúde destes produtos químicos podem estar relacionados à sua persistência e bioacumulação em humanos. As principais vias de exposição são fontes alimentares, inalação e ingestão através de pó, como por exemplo o pó doméstico. Dos retardadores de chama o TBBPA (Tetrabromobisphenol A) é o composto mais estudado devido à sua toxicidade e deteção em diversos meios ambientes e no ser humano. A sua incidência e/ou prevalência de problemas de saúde associados à perturbação endócrina tem aumentado ao longo dos anos. Estudos recentes têm sugerido que o TBBPA contribui para uma série de problemas de saúde, que envolvem não só, doenças como o cancro da mama ou dos rins, mas também incluem doenças metabólicas, como a obesidade. Adicionalmente foram reportados efeitos a nível do desenvolvimento e reprodução humana, assim como a nível da tiroide, sistema cardiovascular e sistema neuro-endócrino. O TBBPA foi identificado em amostras abióticas, como água, ar e poeira, solo, sedimentos e polímeros plásticos, e em amostras bióticas, como soro humano, plasma, urina e leite materno. Além disso, o TBBPA também foi detetado no cordão umbilical de grávidas, comprovando uma exposição pré-natal a este composto. Este resultado sugere que o TBBPA pode atravessar a placenta humana. Então, os efeitos da disrupção endócrina resultantes da exposição ao TBBPA podem ser detetados em gerações futuras? Estudos em embriões e larvas de peixes-zebra, evidenciaram que o TBBPA pode causar toxicidade no sistema cardíaco comprometendo o seu desenvolvimento, resultado do stress oxidativo e consequentemente apoptose celular provocado por este composto. Foi também demonstrado que o TBBPA pode induzir hiperemia e pericardite (edema pericárdico) em embriões e larvas de peixes-zebra. Assim, estes resultados mostraram que há uma relação dose-resposta significativa entre os parâmetros de toxicidade (taxa de eclosão, taxa de sobrevivência, taxa de malformação e taxa de crescimento) e a concentração do TBBPA, nas gerações futuras. A nível vascular, não existem dados publicados relativamente aos efeitos do TBBPA, nem sobre o seu possível papel no desenvolvimento de desordens vasculares. Neste sentido, o trabalho desenvolvido nesta tese de doutoramento teve como principal objetivo o estudo dos efeitos do TBBPA a nível da contratilidade arterial e a análise dos possíveis mecanismos envolvidos nesses efeitos, para futuramente se estudar se o efeito do TBBPA pode ou não estar associado ao desenvolvimento de patologias vasculares, de forma a minimizar o impacto da exposição ao TBBPA nas gerações futuras. Esta análise foi realizada em dois modelos de estudo diferentes, nomeadamente em artérias umbilicais humana (HUA) e em aortas de rato. A HUA é facilmente obtida a partir do cordão umbilical, está implicada na circulação feto-placentária e é uma excelente fonte de células musculares lisas vasculares (VSMC). Mecanismos endócrinos e parácrinos que regulam o estado contrátil das VSMC na HUA são muito importantes para permitir as trocas gasosas e de nutrientes entre o feto e a placenta, uma vez que os vasos sanguíneos do cordão umbilical não são inervados. Estas características torna a HUA um bom modelo para analisar os efeitos dos EDCs no sistema vascular e compreender possíveis implicações vasculares da exposição a esses compostos na gravidez. Em relação à aorta de rato, é um modelo que tem sido utilizado ao longo dos anos devido a uma boa extrapolação dos resultados para o humano. Para além do objetivo principal, anteriormente mencionado, no primeiro trabalho apresentado nesta tese, analisou-se a compartimentação do cGMP (cyclic Guanosine 3,5’- Monophosphate) a nível vascular através da ativação dos CNG (cyclic nucleotide gated channels) sensíveis a cGMP e a possível implicação das fosfodiesterases (PDE). A técnica patch clamp na configuração whole cell foi usada para medir o sinal de ativação dos canais de CNG. As células musculares lisas da artéria umbilical humana (HUASMC) foram infetadas com o adenovírus WT-CNGA2. Os compostos utilizados foram: peptídeo natriurético atrial (ANP), nitroprussiato de sódio (SNP), 3-isobutil-1-metilxantina (IBMX) (inibidor não seletivo das PDE), To-156 (inibidor específico da PDE5), ciloestamida (inibidor específico da PDE3) e Sp-8 (análogo da molécula cGMP). Analisando os resultados obtidos, observa-se que o ANP e o SNP induzem um diferente aumento da ICNG. O sinal do cGMP induzido pelo ANP parece ser controlado pela PDE5 e pela PDE3. Contudo, a administração do SNP parece criar dois efeitos separados, um mais localizado junto à membrana plasmática que é controlado pela PDE3 e pela PDE5, e o outro efeito localizado no interior das células que é regulado apenas pela PDE3. Em suma, a distribuição temporal e espacial diferente do cGMP pode contribuir para efeitos específicos do ANP e de dadores de oxido nítrico (NO) na função vascular, confirmando que a regulação e a síntese do cGMP são compartimentadas nas HUASMC. No segundo trabalho apresentado foram avaliados os efeitos diretos e os efeitos após 24h de exposição ao TBBPA na HUA e analisado o seu possível modo de ação (MOA). O ensaio de MTT foi usado para analisar viabilidade celular das células musculares lisas da artéria umbilical humana (HUASMC), estas células foram expostas a diferentes concentrações de TBBPA, e observou-se uma diminuição da viabilidade celular nas concentrações maiores (500 e 1000 μM). Usando a técnica de banho de órgãos, anéis HUA sem endotélio foram contraídos com serotonina (5-HT), histamina (His) e cloreto de potássio (KCl) e, em seguida, os efeitos diretos do TBBPA (0,01-100 μM) foram analisados. Após 24 horas de exposição ao TBBPA (1, 10 e 50 μM) foram avaliadas as respostas contráteis da HUA à aplicação dos agentes contráteis, 5-HT His e KCl. Para investigar mais detalhadamente o modo de ação vascular do TBBPA, através do qual ele prejudica a homeostase vascular do HUA. Além disso, o mecanismo de ação vascular do TBBPA foi estudado através da análise da atividade dos nucleótidos cíclicos e dos canais de cálcio (Ca2+), vias envolvidas respetivamente, no relaxamento e na contração da HUA. Os resultados obtidos demonstraram que os efeitos diretos do TBBPA induzem um vasorelaxamento da HUA e que a exposição de 24 horas ao TBBPA altera o padrão de resposta vasoconstritora de 5-HT, His e KCl e o padrão de resposta vasorelaxante do SNP e da nifedipina (Nif). Este efeito é devido ao envolvimento do TBBPA com a via NO/sGC/cGMP/PKG e com a interferência no influxo de Ca2+. Além disso, usando a reação em cadeia da polimerase quantitativa em tempo real (qPCR), observou-se que o TBBPA modifica a expressão dos canais de Ca2+ tipo L, das subunidades α- e β1 dos canais de potássio ativados por cálcio (BKCa), da guanilato ciclase solúvel (sGC) e da proteína cinase G (PKG). Assim, estes resultados apontam para alterações na homeostase vascular da HUA provocadas pela exposição ao TBBPA. No terceiro trabalho apresentado foi analisado o efeito do TBBPA no músculo liso da aorta de ratos, para investigar a sua via de sinalização. Para atingir este objetivo, começamos também com a análise da viabilidade celular das células A7r5, usando o ensaio de MTT. As células A7r5 foram expostas durante 24 horas a diferentes concentrações de TBBPA, e os resultados obtidos mostraram que as maiores concentrações de TBBPA (500 e 1000 μM) diminuíram a viabilidade celular. Em seguida, pela técnica do banho de órgãos, os anéis de aorta de rato sem endotélio foram contraídos com Fenilefrina (Phenyl), Noradrenalina (NA) e com uma solução KCl isosmótico para avaliar o efeito vascular do TBBPA (0,01–100 μM). Para além disso, o mecanismo de ação do TBBPA foi estudado através de inibidores específicos, nomeadamente, a Nif, um inibidor de canais de cálcio dependentes de voltagem tipo L, o tetraetilamonio (TEA), um inibidor de canais de potássio dependentes de cálcio (BKCa), a 4-aminopiridina (4-AP), um inibidor de canais de potássio dependentes de voltagem (Kv) e a glibenclamida (Gly), um inibidor de canais de potássio dependentes de ATP (KATP). Os resultados mostraram que estes inibidores reduziram o efeito vasorelaxante do TBBPA, sugerindo que os efeitos vasculares do TBBPA envolvem os canais de Ca2+ e de K+. Para avaliar a atividade dos canais de cálcio dependentes de voltagem (VGCC) tipo L em células A7r5, aplicou-se a técnica patch clamp na configuração de whole cell, e observou-se uma diminuição da corrente de Ca2+. Estes resultados suportam a ideia que os efeitos do TBBPA induzem vasorelaxamento na aorta de ratos, devido à inibição dos canais de Ca2+ e ativação dos canais de K+. Também, por qPCR, observou-se que o TBBPA modula a expressão dos canais de Ca2+ tipo L, das subunidades α- e β1 dos BKCa, da sGC e da PKG. Em suma, os resultados obtidos nesta tese, durante o desenvolvimento deste projeto, confirmaram as ações cruciais do TBBPA no músculo liso vascular. Estes resultados demonstram que o TBBPA induz um relaxamento do músculo liso agindo através de um mecanismo independente do endotélio. Este mecanismo de ação do TBBPA envolve ativação de sGC, o aumento os níveis intracelulares de cGMP, uma inibição dos VGCC tipo L e uma ativação dos canais de K+. Outro resultado inovador da presente tese foi a identificação da existência de compartimentação de cGMP em células musculares lisas vasculares humanas. Devido à alteração da homeostase vascular induzida por TBBPA, este composto pode ser um possível indutor de doenças hipertensivas. Nesta linha de investigação, os resultados obtidos parecem muito promissores, neste sentido estudos adicionais são necessários para conhecer melhor o mecanismo de ação do TBBPA a nível vascular e compreender a sua complexidade na exposição humana e ambiental, de forma a minimizar o risco em gerações futuras

    The Impact of Heterogenous Cell Populations on Impedance-Based Cell Analysis

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    Many in vitro studies for drug development are based on population-averaging measurement techniques without giving information about cell-to-cell variability within the cell ensembles under study. However, such heterogeneities in cell cultures are omnipresent and can arise by several causes, like spontaneous genetic mutations, different metabolic situations or different cell cycle states of individual cells. Moreover, microenvironmental conditions, like cell crowding, might force cell ensembles to form subpopulations with distinct characteristics. Therefore, single phenotypically different subpopulations may be overseen or averaged responses across different subpopulations might not reflect the majority of the cells, leading to misinterpretations of data – one possible reason for the high failure rate in clinical trials. This thesis addressed the fundamental question of how cell-to-cell variability in populations influence the signal of population-based assays by using three different approaches. The first project addressed the impact of evenly distributed heterogeneities within cell populations, introduced by mixing a cell line expressing a certain G protein-coupled receptor (GPCR) with a cell line not expressing this receptor type, on the impedance-based cell analysis. The second project focused on the development of an impedance-based assay for the future purpose of spatiotemporally introducing heterogeneities in an isogenic cell population, expressing a certain GPCR, by switching an appropriate, photochromic ligand by illumination. The third project addressed the quantification of the impact of heterogeneities within cell populations on the impedance-based cell analysis in a theoretical manner. The first project focused on the impact of cell-to-cell variability on the population-based impedance signal by mixing cell lines in different ratios prior to the seeding onto the co-planar gold electrodes. The evenly distributed heterogeneities in the resulting cell populations were generated by co-culturing two cell lines with one of them expressing a GPCR predominantly coupled to one of the three main canonical G-protein pathways (Gq, Gs, Gi/o). A protocol was established to obtain co-cultures with distinct cell ratios resulting in well-defined areal receptor densities (ARD) as verified by supported microscopic staining studies. The stimulation of cell ensembles with varying ARD by the GPCR's endogenous ligands was analyzed in detail by wholistic impedance-based cell assays. Efficacies and potencies, which describe the maximal agonist effect and the activity of a drug, were compared to those of the pure and original cell lines. It was shown that both parameters were dependent on the ARD and the coupled signaling cascades in distinct ways: for the Gq pathway, efficacy decreased non-linearly with decreasing ARD, while the Gs- and Gi/o-pathways exhibited an almost linear dependency of efficacy on the ARD. The potencies observed for the Gq- and Gi/o-coupled signaling pathway decreased with decreasing ARD, while the potency of the Gs-pathway was almost independent of the ARD. Simple simulations indicated that underlying communication processes between stimulated and non-stimulated cells within the populations under study may be responsible for these trends. Additionally, two proximal assay techniques were used to assist the interpretation of impedance analysis and to assign the impact of the ARD on the signal to a certain part of the signaling cascade. The radioligand competition binding assay confirmed the correct co-culturing strategy for such heterogeneous cell populations and confirmed the corresponding potency to be independent of the population composition. Population-based Ca2+ imaging highlighted the impact of altering the ARD on second messenger mobilization. Again, the ARD did not affect the potency, but the analysis of the response on a single-cell level proposed cell communication as a potential mechanism explaining the dependency of impedance on ARD. Moreover, the stimulation of a co-culture, consisting of two GPCR-expressing cell lines, was analyzed impedimetrically. The outcomes indicated that the potency dependency on the ARD was caused by the simultaneous activation of two different signaling pathways. The obtained data confirmed that the impact of artificially introduced heterogeneities in the cell population under study on the obtained impedance signal was indeed significant. Nevertheless, it remains elusive, whether these results can be translated to other cell lines or other GPCRs. This project addressed the fundamental question of areal heterogeneities influencing the impedance signal. However, further studies on cell ensembles with different compositions and other measurement techniques have to be carried out to obtain a broader picture of such impacts on population-based measurements and its significance for the drug development process. In the second project of this thesis, an assay was developed for the future purpose of introducing cell-to-cell variability within isogenic cell populations by spatiotemporal illumination of photochromic GPCR-ligands, which can be toggled between their bioactive and -inactive isomer. Thus, it was required to establish a protocol to active in situ such a ligand by online irradiation with light and to monitor the cell responses in a time-resolved manner. The wholistic impedance-based cell assay was appropriate to monitor the in situ toggling of a model photoswitchable ligand for a Gq-coupled receptor. To accomplish the superordinate goal, it will be necessary to establish a measurement setup, which is capable of spatiotemporal illumination of the cell culture, so that a small subpopulation can be stimulated in a spatiotemporally well-defined manner after the systemic addition of the bioinactive species of a photoswitchable ligand. The third part of this thesis addressed the impact of heterogeneous cell populations on the impedance readout by theoretical means. For this purpose, a MATLAB-based algorithm was developed, capable of simulating different cell types following the electric cell-substrate impedance sensing (ECIS) model. In contrast to the conventional mode, which assumes global cell-related parameters (α for the cell-substrate contacts, Rb for the cell-cell contacts, Cm for the cell membrane capacitances) for the whole population, the new approach emulated cell populations by cell-related parameters, each showing a Gaussian distribution with a mean and a deviation value. After successful validation of the underlying algorithm, discrepancies from the ECIS model using global parameters were found for such populations with heterogeneous cell-related parameters for three distinct cell types, emulating leaky, moderately tight, and tight cells. Especially the deviation of the Gaussian-distributed parameters α and Rb had a big impact on the spectra. In direct comparison to the reference, which was a homogenous cell population with global parameter values being equal to the mean values of the Gaussian distribution, a systematical misestimation could be found for α (up to 110 % of the reference value) and underestimation for Rb (down to 78 % of the reference value) when the deviation values were set to 30 % of the mean values. In contrast, Cm was found to be very robust for deviations up to 30 % (100 % of the reference value). In summary, the thesis has demonstrated in an experimental and theoretical manner that cell-to-cell variability has indeed major impacts on the population-based impedance signal, having the potential to misdirect data interpretation. These can affect fundamental as well as pharmacological research. Thus, it is crucial to address such heterogeneities within cell populations in future studies using population- as well as single-cell-based assay techniques

    Subcellular location defines GPCR signal transduction

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    Intracellular G protein-coupled receptors (GPCRs) can be activated by permeant ligands, which contributes to agonist selectivity. Opioid receptors (ORs) provide a notable example, where opioid drugs rapidly activate ORs in the Golgi apparatus. Our knowledge on intracellular GPCR function remains incomplete, and it is unknown whether OR signaling in plasma membrane (PM) and Golgi apparatus differs. Here, we assess the recruitment of signal transducers to mu- and delta-ORs in both compartments. We find that Golgi ORs couple to Gαi/o probes and are phosphorylated but, unlike PM receptors, do not recruit β-arrestin or a specific Gα probe. Molecular dynamics simulations with OR–transducer complexes in bilayers mimicking PM or Golgi composition reveal that the lipid environment promotes the location-selective coupling. We then show that delta-ORs in PM and Golgi have distinct effects on transcription and protein phosphorylation. The study reveals that the subcellular location defines the signaling effects of opioid drugs

    Molecular modeling of the monoamine transporters and their interactions with psychostimulants and other substances

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    The monoamines (i.e., dopamine, serotonin, and norepinephrine) are vital to the ontogeny, function, and plasticity of the nervous system. These neurotransmitters affect each other and regulate, amongst others, motor function, cognitive state, motivation, and stress reactions. The neurotransmission is mainly terminated by reuptake in monoamine transporters (MATs), i.e., the dopamine-, serotonin-, and norepinephrine transporter. These transporters are the focus of the current study. Imbalance in the monoamine systems in the central nervous system (CNS) is associated with neurological- and psychiatric disorders, where the MATs are targets for several therapeutic drugs. Most of these drugs bind the outward-facing conformation of the MATs, and their effects depend highly on the selectivity for a single MAT. On the other hand, the increased use of illicit stimulants, predominantly acting on DAT, has risen alarms due to their unpredictable effects and high abuse potential. Regarding this, some research standards (atypical inhibitors), suggested to bind the inward-facing conformation of the MATs, have been shown to exert anti-addictive properties – being valuable in future treatment of addiction and withdrawal symptoms. The main aim of this thesis was to construct outward-and inward facing human MAT-models, based on homology modeling, to identify determinants for selective binding to each MAT, by utilizing induced fit docking and molecular dynamics simulations. Therapeutic psychostimulants, illicit psychostimulants, antidepressants, non-stimulants, atypical inhibitors, and some research standards were studied. The results indicate that divergent residues in the S1-site play a key role in MAT-selectivity. These residues shape the polarity and steric environment in the orthosteric (S1) pocket, thus affecting the stabilization, interactions, and orientation of ligands in each MAT. Structural features in the ligands appeared to also play a role in the selectivity for a MAT, concerning the binding mode and formation of interactions
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