Tesis sobre el tema "Adenosine A2B receptor"

Starting from chemical structure of N-benzo-[1,3]dioxol-5-yl-2-[5-(2,6dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol3-yloxy]-acetamide, MRE2029F20* various structural modifications were realized to afford a new series of A2B antagonists. The bioisosteric replacement of the anilide moiety with benzimidazole or quinazoline rings, the effect of the substitution of pyrazole with isoxazole moiety were investigated. Amide bond has been also replaced with the 5phenyl-1,2,4-oxadiazole nucleus on the basis of other adenosine pharmacophores reported previously. In this context the effect of the nitrogen at the 9-position has been also studied preparing four 9-deaza direct analogs of 8pyrazol-xanthine compounds to compare affinity and selectivity at A2B adenosine receptor. The most significant result was obtained by bioisosteric replacement of the anilide moiety with benzimidazole, achieving antagonists with high affinity and selectivity toward the A2BAR. In particular compound 8-[5-(4-Chloro-6-trifluoromethyl-1H-benzoimidazol-2-ylmethoxy)-2methyl-2H-pyrazol-3-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (hA1 Ki = 2530 nM, hA2A Ki > 1000 nM, hA2B Ki = 9.4 nM, hA3 Ki > 1000 nM) and compound 8-[5-(4,6-Bis-trifluoromethyl-1H-benzoimidazol-2-ylmethoxy)-2methyl-2H-pyrazol-3-yl]-1,3-dipropyl-3,7 dihydro-purine-2,6-dione (hA1 Ki = 4462 nM, hA2A Ki > 1000 nM, hA2B Ki = 25 nM, hA3 Ki > 1000 nM), showed the best biological data. These new selective and potent A2B antagonists will aid in the elucidation of the physiological role of this receptor and possibily lead to therapeutilally useful agents for treating asthma, diabetes and other diseases.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Cardiovascular disease remains a leading cause of mortality. Risk factors, including poor glycemic control, central obesity and dyslipidemia contribute to prothrombotic and proinflammatory states, which elevate the risk for adverse cardiovascular events. Identifying new pharmacological targets for glycemic control is essential for prevention and management of cardiovascular disease. Adenosine is an endogenous purine nucleoside released from various tissues following stress, or produced externally by ecto-nucleotidases. Adenosine modulates inflammation and influences the metabolic state. There are four adenosine receptors classified as adenylyl cyclase activating (A2a and A2b) or inhibiting (A1 and A3). Using an A2b adenosine receptor (A2bAR) knockout (KO) mouse, our laboratory previously showed that A2bAR protects against atherosclerosis. Subjecting A2bAR, Apolipoprotein E (ApoE) double KO mice to a high fat diet (HFD) led to augmented liver levels of the transcription factor sterol response element binding protein-1 (SREBP-1) and its downstream targets, cholesterol and triglycerides, as well as to increased atherosclerosis, compared to mice with normal A2bAR. The studies in this thesis showed that selective restoration of hepatic A2bAR by adenovirus mediated gene transfer, or in vivo administration of an A2bAR agonist, BAY 60-6853, reduced the lipid profile and atherosclerosis. This study identified the A2bAR as a therapeutic target for hyperlipidemia and atherosclerosis. Liver steatosis is often associated with impaired hepatic insulin signaling. To test the hypothesis that A2bAR controls glucose/insulin homeostasis, A2bAR KO mice, with normal ApoE background, were subjected to HFD. Compared to control mice, A2bAR KO mice developed obesity and hallmarks of type 2 diabetes (T2D). We identified a novel link between expression of A2bAR and insulin receptor substrate 2 (IRS-2). IRS-2 is downregulated and insulin signaling is impaired in tissues of A2bAR KO mice that exhibit a greater inflammatory state. Importantly, pharmacological activation of A2bAR under HFD, using BAY 60-6583, restored IRS-2 levels, and ameliorated T2D. In obese human subjects, A2bAR expression correlated strongly with IRS-2 expression. Taken together, our study identifies the A2bAR as a significant regulator of HFD-induced hallmarks of atherosclerosis and T2D, with dysregulated liver A2bAR expression being a common denominator. Our study points to A2bAR as a therapeutic target for these disorders.

Thesis (Ph.D.)--Boston University
The development, maintenance and repair of the skeletal system are dependent on the differentiation of both chondrocytes and osteoblasts from their common progenitor, the mesenchymal stem cell (MSC). The A2B adenosine receptor (A2BAR) is a G-protein-coupled receptor that signals by increasing cAMP and/or activating phospholipase C signaling. Considering the published roles of cAMP on MSC differentiation, and our finding that the expression of the A2BAR is induced following injury, we hypothesized that ablation or activation of the A2BAR impacts the differentiation of osteoblasts and chondrocytes and that this would manifest as changes in skeletal development and bone fracture repair. Activation of the A2BAR increased the differentiation of bone marrow-derived MSCs to osteoblasts by increasing mRNA expression of the transcription factors runt-related transcription factor 2 (Runx2) and Sp7 transcription factor (Osterix), which are essential for osteoblast differentiation. To examine the effect of the A2BAR on bone formation in vivo, we subjected wild type (WT) and A2BAR knockout (KO) mice to bone fracture. A2BAR KO mice had impaired bone formation during fracture repair with increased cartilage volume. As fracture repair recapitulates the events that occur during endochondral ossification, we compared the growth plates of WT and A2BAR KO mice. In comparison to WT, A2BAR KO mice had a shorter growth plate initially, but a taller growth plate at a later age. These results suggest that initiation of endochondral ossification may be delayed in the A2BAR KO mice. Finally, we investigated whether the A2BAR is involved in chondrocyte differentiation. A2BAR activation decreased mRNA expression of the key transcription factor for chondrocyte differentiation, SRY (sex-determining region Y)-box 9 (Sox9) and decreased the mRNA expression of the hypertrophic chondrocyte marker Collagen X. Taken together, these data demonstrate a previously unidentified role of the A2BAR receptor in regulating MSC differentiation to both osteoblast and chondrocyte lineages. Further, we showed that mice null for the A2BAR have dysregulated bone formation during development and after injury. The importance of this receptor during bone formation and fracture repair could have implications for A2BAR-based therapies for bone maintenance and repair.

2016 - 2017
The adenosinergic pathway plays a critical role in cancer development and progression, as well as in drug resistance to chemotherapy and/or targeted-therapy. The goal of this PhD thesis was to investigate and fully characterize the role of CD73/adenosine A2A-A2B receptors axis in cancer, highlighting the therapeutic potential of inhibitors of the adenosinergic pathway. We firstly characterized the mechanism/s by which A2BR promotes immunosuppression and angiogenesis in tumor-bearing hosts, focusing on the role of myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs). The results revealed that treatment of melanoma-bearing mice with Bay60-6583, a selective A2BR agonist, is associated with 1. increased tumor VEGF-A expression and vessel density, and 2. increased accumulation of tumor-infiltrating CD11b+Gr1+cells (MDSCs). MDSCs strongly contribute to the immunosuppressive and angiogenic effects of Bay60-6583. Melanoma-bearing mice treated with a selective A2BR antagonist PSB1115 showed reduced tumor growth compared to controls and this effect was associated with reduced tumor angiogenesis, low levels of MDSCs and increased number of tumor-infiltrating CD8+ T cells. Furthermore, blockade of A2BR increased the anti-tumor effects of VEGF-A inhibitors. Next, we verified that A2BR activation also drives fibroblasts activation within melanoma tissues, by increasing the number of FAP positive cells within tumor lesions. FAP is a common marker of activated fibroblasts also named cancer-associated fibroblasts. These cells produce and secrete various tumor-promoting factors, including fibroblast growth factor (FGF)-2 and CXCL12 or stromal-derived factor 1 α (SDF1α), that were increased both in melanoma tissue and fibroblasts isolated from melanoma tissue or from skin upon Bay60-6583 treatment. Bay60-6583-induced FGF-2 from fibroblasts contributed to melanoma cells proliferation. The CXCL12/CXCR4 pathway, instead, was involved in the pro-angiogenic effects of A2BR agonist, but not in its immunosuppressive effects. These effects were significantly blocked by the A2BR antagonists PSB1115. Taken together, these data elucidate the pivotal role of A2BR in establishing a positive cross-talk between tumor-infiltrating immune cells, fibroblasts and endothelial cells that sustain tumor growth, reinforcing the therapeutic potential of A2BR blockers for cancer therapy. ... [edited by Author]
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A hipertensão arterial é um problema de saúde pública no Brasil, pois aproximadamente 20 % da população adulta desenvolve hipertensão essencial, cujas causas ainda não são conhecidas. No entanto, sua gênese pode estar relacionada com disfunção nas áreas do sistema nervoso central (SNC) que regulam o sistema cardiovascular. O núcleo do trato solitário (NTS) e o bulbo ventrolateral são áreas importantes no controle neural da pressão arterial. Os receptores de adenosina A2a (rA2a) são encontrados em todo o SNC e estão relacionados com estudos terapêuticos de diversas doenças. No NTS a estimulação dos rA2a provoca ajustes pontuais em outros sistemas de neurotransmissão, além de diminuir a pressão arterial. A nicotina é uma molécula com uma vasta faixa de efeitos modulatórios em nosso organismo. Entre esses efeitos se destacam a capacidade de interagir com diversos sistemas de neurotransmissão nas áreas do bulbo relacionadas com a regulação da pressão arterial e de antecipar e/ou intensificar o desenvolvimento da hipertensão em sujeitos com pré-disposição genética. Desta forma, o objetivo do presente trabalho é avaliar o efeito modulatório da nicotina sobre o rA2a em cultura mista de neurônios e células gliais da porção dorso-medial do bulbo de ratos geneticamente hipertenso (SHR) e normotensos (WKY). Para isso, utilizaram-se técnicas como a de PCR em tempo real, Western Blotting e análise de ligação do receptor. Nossos resultados demonstraram que: 1) em condição basal células de ratos normotensos apresentam maior ligação do rA2a do que células de ratos hipertensos; 2) tratamento com nicotina resultou na diminuição da ligação do receptor em ambas as cepas, com um efeito de maior magnitude em células de ratos WKY; 3) nas duas linhagens o tratamento com nicotina alterou os níveis protéicos do rA2a, assim como o RNAm do receptor; 4) a linhagem e o tratamento separadamente, como a interação entre ambos influenciaram na expressão do RNAm , níveis protéicos e ligação do rA2a nas células dos ratos WKY e SHR. Por fim, os resultados apresentados aqui indicam que o rA2a em células de ratos hipertensos tem sua função deprimida em comparação com as células de ratos normotensos; e que a nicotina foi capaz de modular o funcionamento do rA2a, o qual pode influenciar no controle da pressão arterial. Esses dados são bastante interessantes, pois abrem novas perspectivas de análise dos mecanismos intracelulares envolvidos na modulação dos rA2a pela nicotina, assim como a importância desse sistema no desenvolvimento da hipertensão
Hypertension is one of the most common worldwide diseases afflicting humans. Because of the associated with morbidity and mortality and the cost to the society, it became an important public health challenge in Brazil. The mechanisms involved in development of hypertension still remain unclear However, hypertension can result from neuronal network imbalance in areas of the central nervous system that control blood pressure. The nucleus tractus solitarius (NTS) plays an important role in cardiovascular control. Within the NTS there are several neurotransmitters and neuromodulatory substances, such as adenosine, which acts on purinoreceptors A2a (A2ar). The A2ar modulates neurotransmission in the NTS and its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that cross the blood-brain barrier and acts in several areas of central nervous system including the NTS. In this nucleus, nicotine is able to interact with some neurotransmitter systems and contributes for the development of hypertension in subjects with genetic predisposition to this disease. The goal of this study was to analyze the modulatory effects of nicotine on A2ar in cultured neurons and glial cells from medulla oblongata of normotensive (WKY) and spontaneously hypertensive rats (SHR). By means of real time PCR, Western Blotting and binding receptor assay. We have demonstrated that in basal condition cells of WKY presents increased binding of A2ar than the cells of SHR. Nicotine treatment induced a decrease in the binding of A2ar in both strains, however, this response was more pronounced in cells of WKY than SHR. Changes in mRNA and protein levels of A2ar was also observed in response to nicotine treatment. The strains and treatment separately, as well as the interaction between them influenced mRNA expression, protein level and binding of A2ar in NTS cells of WKY and SHR rats. Finally, these results show for the first time changes in A2ar mRNA expression, protein level and binding in cells from the medulla oblongata of WKY and SHR rats, as well as, the nicotine modulation upon this system, which might influence cardiovascular control. These data open up new approaches for the study of intracellular mechanisms involved in the modulation of adenosine A2a receptor by nicotine, as well as the importance of this interaction in the development of hypertension.

Los receptores acoplados a proteína G (GPCR) conforman la familia de receptores de membrana más grande. El numeroso y variado tipo de señales que detectan han otorgado a estos receptores un alto interés farmacológico. Además, las interacciones entre diferentes tipos de GPCR formando complejos oligoméricos dan lugar a complejos con características bioquímicas diferenciadas de los protómeros que los forman. En esta Tesis Doctoral se han estudiado diferentes aspectos derivados este tipo de interacciones, centrando estos experimentos alrededor del receptor A2A de adenosina (A2AR), un importante neuromodulador del Sistema Nervioso Central. Por una parte, mediante la combinación de las técnicas de transferencia de energía resonante bioluminiscente (BRET) y de complementación bimolecular fluorescente (BiFC) se ha podido detectar in vivo que A2AR forma oligómeros con más de dos protómeros, tanto de tipo homomérico (Gandía et al., 2008) como heteromérico. En este último caso, se ha estudiado en concreto el oligómero de A2AR con el receptor D2 de dopamina y el receptor metabotrópico 5 de glutamato (Cabello et al., 2009). A continuación, se ha aplicado una variante de la técnica de doble híbrido específica para proteínas de membrana (MYTH), con la intención de detectar proteínas interaccionantes con A2AR. Gracias a esta aproximación, se han encontrado nuevas proteínas candidatas a interaccionar con nuestro receptor, destacando entre ellas un GPCR huérfano, GPR37. Mediante técnicas físicas y funcionales en modelos de cultivo celular y animales se ha podido validar la interacción A2AR/GPR37 y se ha comprobado que la presencia de GPR37 modifica la funcionalidad del receptor A2A de adenosina. Finalmente, para profundizar en las características estructurales de GPR37, poco conocidas hasta el momento, se ha estudiado la cola C-terminal del receptor. Así, se ha visto que existe una región rica en residuos de cisteína que regula el tráfico del receptor hacia la membrana plasmática. Además, este dominio rico en cisteínas modula el estrés de retículo endoplasmático generado al sobreexpresar GPR37 en cultivo celular y también la inducción de vías apoptóticas (actividad de caspasa-3) en estas mismas condiciones (Gandía et al., 2013).
G protein-coupled receptors (GPCR) consitute the biggest family of membrane receptors. Since they detect a large and diverse number of signals, they have a growing pharmacological interest. Furthermore, the interactions between different types of GPCR form oligomeric complexes that show different biochemical properties than the protomers they are made of. Different aspects of these interactions have been studied in this Doctoral Thesis, focusing the experiments around the adenosine A2A receptor, being adenosine an important modulator of the Central Nervous System. Firstly, by means of the combination of the bioluminescent ressonant energy transfer (BRET) and bimolecular fluorescent combination (BiFC) techniques we have detected in vivo that A2AR is able to form oligomers made up of more than two protomers, leading to homomeric complexes (Gandía et al., 2008) as well as others of heteromeric nature. In this latter case, we have studied the oligomer of A2AR with the dopamine D2 and glutamate metabotropic 5 receptors (Cabello et al., 2009). Following these experiments, we have applied a modified version of the yeast two-hybrid technique set up for membrane proteins (MYTH) in order to detect A2AR-interacting proteins. Thanks to this approach, we have found new potential interactors, and among them an orphan GPCR has stood out: GPR37. By means of physical and functional techniques in cell culture and animal models we have validated the A2AR/GPR37 interaction and we have demonstrated that the presence of GPR37 modifies the functionality of A2AR. Finally, in order to better understand the rather less studied structural characteristics of GPR37, we have studied its C-terminal tail. Thus, we have observed the presence of a cysteine-rich region that regulates the trafficking of the receptor to the plasma membrane. Furthermore, this cystein-rich domain modulates the GPR37-dependent endoplasmic reticulum stress, as well as the induction of apoptotic pathways (capase-3 activity) (Gandía et al., 2013).

The adenosinergic system is a neuromodulatory complex acting on four receptor subtypes with varying distributions throughout the central nervous system. This system contributes to homeostasis and regulates several physiological functions including sleep, motor function, synaptic plasticity or cognition. Adenosine can exert an upstream modulation of several neurotransmitter systems, including dopamine and glutamate, both implicated in the pathophysiology of neuropsychiatric diseases and particularly in psychosis. Schizophrenia is considered a psychotic disease characterized by a chronic and debilitating course that can be triggered by different genetic, developmental and environmental factors. The disease is manifested with three symptom domains including positive, negative and cognitive symptoms and has important treatment limitations. Preclinical and clinical data suggest that adenosine dysfunction may contribute to the pathophysiology of schizophrenia. Hence, the main focus of this thesis is to study the responses induced by the complete deletion of adenosine A2A receptors in the pathophysiology of the disease, paying special attention to the cognitive symptom domain. The use of biochemical and behavioural approaches has allowed us to assess the validity of A2A receptor KO as a possible animal model for the study of schizophrenia.
El sistema adenosinèrgic és un neuromodulador que actua sobre quatre subtipus de receptors amb diferents distribucions en el sistema nerviós central. Aquest sistema contribueix a l’homeòstasi i a la vegada regula diverses funcions fisiològiques com per exemple la son, la funció motora, la plasticitat sinàptica o la cognició. L’adenosina pot exercir un control “upstream” sobre diferents sistemes de neurotransmissió, incloent la dopamina i el glutamat, neurotransmissors implicats en la fisiopatologia de les malalties neuropsiquiàtriques, especialment en la psicosis. L’esquizofrènia es considera una malaltia psicòtica que cursa de forma crònica i debilitant provocada per la interacció de diferents factors genètics, ambientals i del desenvolupament. La malaltia es manifesta en tres dominis de símptomes, incloent símptomes positius, negatius i cognitius, i actualment té importants limitacions terapèutiques. Estudis preclínics i clínics suggereixen que la disfunció en el sistema d’adenosina pot contribuir a la patofisiologia de l’esquizofrènia. Per tant, l’objectiu principal d’aquesta tesi és l’estudi dels efectes induïts per la supressió completa dels receptors A2A d’adenosina en la fisiopatologia de la malaltia, parant especial atenció al domini de símptomes cognitius. L’ús de mètodes bioquímics i comportamentals ens ha permès avaluar la validesa dels KO pel receptor A2A d’adenosina com a possible model animal per a l’estudi de l’esquizofrènia.

G-protein coupled receptors (GPCRs) are one of the main targets for drug design. GPCR pharmaceutical research has bottlenecked on drug research, and the current assay systems are not creating therapeutic drugs from drug hits. The principal aim of this project was to establish a relationship between drug affinity (PKi) and drug intrinsic efficacy (Emax) using receptor conformational change parameters (measurements defined by fluorescence resonance energy transfer (FRET) experiments) to provide a new way of screening for the intrinsic efficacy of a drug. This project aimed to develop a drug screen based on the hypothesis that ligand intrinsic efficacy was linked to ligand induced receptor conformational change. Using FRET technology, the extent and rate of ligand induced receptor conformational changes were investigated. In contrast to the clear cut nature of ligand intrinsic efficacy and the extent of ligand induced receptor conformational change observed in the literature no correlation was found in this project on the FRET reporter construct A2A.4C.CFP. FRET studies revealed some key findings into the receptor activation mechanisms of the A2aR. Firstly, that different agoinsts induce different conformational states of the receptor and at different rates, thus these finding support activation models proposed by Koshland and are inline with work carried out by Kobilka. Secondly, that different agonists imposed different conformational restrictions/conditions on the resultant conformation of the second agonist applied. This finding opens up the possibility for drug design, as it is possible that the resultant conformation of a drug applied after the receptor has been activated by a previously applied agonist may result in a different pharmacological response, and hence may open up new avenues for investigation. cAMP functional studies were carried out to classify and rank the intrinsic efficacy of the test ligands and to assess effects of the mutations within the A2A.4C.CFP receptor on downstream signalling. All the ligands tested with the exception of compound 15 were reported to be full A2aR agonists in the literature. However compounds CV 1808 and 2Chloroadenosine both generated only partial responses in this assay. This finding may be reflective of differences in receptor activation mechanism of these two compounds compared to the other full agonists.

A cafeína é o psicoestimulante mais consumido mundialmente. Sua ação farmacológica consiste em bloquear os receptores de adenosina A1 e A2A. A administração crônica de cafeína previne déficits cognitivos decorrentes da idade e em modelos experimentais da doença de Alzheimer. Esses efeitos preventivos são também observados pela administração do antagonista seletivo do receptor de adenosina A2A. Nesse trabalho investigou-se a participação dos receptores de adenosina A1 e A2A na prevenção dos déficits cognitivos induzidos por escopolamina. Também foi investigado se a manipulação dos receptores A2A teria algum impacto na memória em camundongos naive. Para isso, foram utilizadas três tarefas comportamentais que avaliaram três tipos de memória: a tarefa de reconhecimento de objetos (RO), a esquiva inibitória (EI) e o labirinto em Y. A administração intraperitoneal de escopolamina (1,0 mg/kg) prejudicou o desempenho da memória de curto prazo nas três tarefas utilizadas. O antagonista de receptores A1 (DPCPX, 1,0 mg / kg, i.p.) preveniu a amnésia induzida por escopolamina no RO e na EI, enquanto o antagonista de receptores A2A (SCH 58261, 0,5 mg / kg, i.p.) preveniu a amnésia em todos os testes. Além disso, ambos os antagonistas não apresentaram efeitos sobre a memória ou a locomoção em animais naive. Também foi observado que a ativação dos receptores A2A, a partir da administração de CGS 21680 (0,1 mg/kg, i.p.) antes da sessão de treino, foi suficiente para provocar prejuízos na memória em animais naive também nas três tarefas, e este efeito foi prevenido por meio da administração de SCH 58261 (0,5 mg/kg, i.p.). Por fim, a administração intracerebroventricular (i.c.v) de CGS 21680 (50 nmol) também prejudicou o desempenho dos animais na tarefa de RO. Em conjunto, estes resultados sugerem que a ativação dos receptores A2A é suficiente para provocar déficits de memória e ainda sugerem que os receptores A1 também participam de maneira seletiva no controle dos déficits de memória relacionados ao sistema colinérgico.
Caffeine, a non-selective adenosine receptor antagonist, prevents memory deficits, an effect mimicked by adenosine A2A receptor (A2AR), but not receptor A1 (A1R), antagonists upon aging and Alzheimer´s disease. We tested if A2AR were also necessary for the memory impairment upon direct perturbation of the cholinergic system with scopolamine and if A2AR activation was sufficient to trigger memory deficits in naive mice using 3 tests, to probe for short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. The intra-peritoneal (i.p.) administration of scopolamine (1.0 mg/kg) impaired short-term memory performance in 3 tests, namely the object recognition task, inhibitory avoidance and modified Y-maze. The scopolamine-induced amnesia was prevented by the A2AR (SCH 58261, 0.5 mg/kg, i.p.) as well as by A1R antagonist (DPCPX, 1 mg/kg, i.p.) in all tests, except for the modified Y-maze, and both antagonists were devoid of effects on memory or locomotion in naive rats. Notably, the activation of A2AR with CGS 21680 (0.1 mg/kg, i.p.) before the training session was sufficient to trigger memory impairment in the 3 tests in naive mice, and effect prevented by SCH 58261 (0.5 mg/kg, i.p.). Furthermore, the intracerebroventricular administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. These results show that A2AR are necessary and sufficient to trigger memory impairment and they further suggest that A1R might also be selectively engaged to control the cholinergic driven memory impairment.

Over 50% of clinically-marketed drugs target membrane proteins; in particular G protein-coupled receptors (GPCRs). GPCRs are vital to living cells, performing an active role in many processes, making them integral to drug development. In nature, GPCRs are not sufficiently abundant for research and their structural integrity is often lost during extraction from cell membranes. The objectives of this thesis were to increase recombinant yield of the GPCR, human adenosine A2A receptor (hA2AR) by investigating bioprocess conditions in large-scale Pichia pastoris and small-scale Saccharomyces cerevisiae cultivations. Extraction of hA2AR from membranes using novel polymers was also investigated. An increased yield of hA2AR from P. pastoris was achieved by investigating the methanol feeding regime. Slow, exponential feed during induction (μlow) was compared to a faster, exponential feed (μhigh) in 35 L pilot-scale bioreactors. Overall hA2AR yields were increased for the μlow cultivation (536.4pmol g-1) compared to the μhigh148.1 pmol g-1. hA2AR levels were maintained in cytotoxic methanol conditions and unexpectedly, pre-induction levels of hA2AR were detected. Small-scale bioreactor work showed that Design of Experiments (DoE) could be applied to screen for bioprocess conditions to give optimal hA2AR yields. Optimal conditions were retrieved for S. cerevisiae using a d-optimal screen and response surface methodology. The conditions were 22°C, pH 6.0, 30% DO without dimethyl sulphoxide. A polynomial equation was generated to predict hA2AR yields if conditions varied. Regarding the extraction, poly (maleic anhydride-styrene) or PMAS was successful in solubilising hA2AR from P. pastoris membranes compared with dodcecyl-β-D-maltoside (DDM) detergent. Variants of PMAS worked well as solubilising agents with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or cholesteryl hemisuccinate (CHS). Moreover, esterification of PMAS improved solubilisation, suggesting that increased hydrophobicity stabilises hA2AR during extraction. Overall, hA2AR yields were improved in both, P. pastoris and S. cerevisiae and the use of novel polymers for efficient extraction was achieved.

Recently, several studies have demonstrated that G protein coupled receptors exist as homo/heterodimers or oligomers. Adenosine A2A receptors and dopamine D2 receptors are present as both homo- and heterodimer. In the GABAergic striatopallidal neurons A2AR are co- localized with D2 receptors (D2R), and establish functional A2AR-D2R heteromers, which modulates dopaminergic activity. Due to be involved in physiological processes, these receptors bear critical roles. Dopamine receptors play critical role in dopaminergic pathways in regulation of memory, food intake and psychomotor activity, etc. On the other hand, adenosine A2A receptors are involved in the regulations of neurotransmission, immune response and cardiovascular systems. Dopamine D2R andadenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity The purpose of this study is to assess the functionality of EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) tagged adenosine A2A and dopamine D2 receptors and to detect homo/ hetero-dimerization of these receptors in live cells via Fluorescence Resonance Energy Transfer (FRET). Understanding the mechanisms of the interaction between adenosine and dopamine signaling will help us to figure out some molecular mechanism of neurophysiological disorders. Furthermore, the fluorescence based live cell model could be used to observe the effects of potential anti-psychotic drugs on the interaction of these two receptors.

To investigate adenosine-opioid-dopamine interactions in addictive processes, in vivo microdialysis and quantitative autoradiography were carried out using mice deficient in the adenosine A2A receptor (A2aR-/-).

Myocardial fibrosis contributes to the pathogenesis of diverse forms of cardiac hypertrophy. Recent evidence suggests a crucial role for purinergic signalling in the formation of tissue fibrosis. In particular, inhibition of adenosine A2A receptors (A2AR) in animal models for hepatic and skin fibrosis resulted in a reduction of fibrosis formation. The specific hypothesis of this investigation was that myocardial stress results in increased interstitial levels of adenosine, which activate cardiac fibroblasts via adenosine A2AR. Accordingly, the specific objective of this investigation was to delineate the role of adenosine A2AR signalling in the development of myocardial fibrosis. In vitro, isolated cardiac fibroblasts demonstrated a significant increase in collagen secretion into CCM upon specific A2AR stimulation. This stimulatory effect was inhibited by the addition of a specific A2AR inhibitor. In vivo, models for cardiac hypertrophy including the transgenic cardiac actin E99K hypertrophic cardiomyopathy model, and a transverse aortic constriction (TAC) model of pressure overload, were investigated as murine models for myocardial fibrosis (MF). In cardiac actin E99K hearts the occurrence of cardiac fibrosis was associated with ventricular dysfunction, as well as energetic perturbations. In addition, a direct linear correlation between myocardial collagen content and interstitial adenosine levels was found in actin E99K hearts upon microdialysis experiments. Crossbreeding of actin E99K and A2AR knock-out (KO) mice resulted in a significant reduction of myocardial collagen content and fibrosis in E99K heterozygous A2AR KO animals compared to E99K heterozygous A2AR wild-type (WT) animals. Further, adenosine A2AR KO mice undergoing transverse aortic constriction demonstrated significantly less fibrosis formation compared to constricted WT mice. This was associated with a significant rescue of cardiac function. In addition, pharmacologic adenosine A2AR inhibition using the antagonist ZM241385 demonstrated a partial rescue of myocardial fibrosis in both TAC and E99K animals. These data suggest that adenosine the A2AR plays a crucial role in the formation of myocardial fibrosis in a variety of cardiac pathologies and that this pathway is susceptible to pharmacologic modulation. A2AR manipulation may contribute to further understanding of pathophysiological pathways in the development and progression of cardiac disease and represents an excellent therapeutic target for clinically available A2AR antagonists.

Background and rationale - Parkinson’s disease is a neurodegenerative disorder characterised by reduced levels of dopamine in the brain. The cause of Parkinson's disease is still unknown; however several theories pertaining to the etiology exist. Current treatment mainly aims at dopamine replacement, with agents such as levodopa and dopamine agonists that provide patients with symptomatic relief. This relief is unfortunately only temporary as the progression of the disease is not halted. Furthermore, these therapies are associated with a range of side effects and novel approaches to the treatment are thus urgently required. Adenosine A2A receptor antagonists recently emerged as a promising non-dopaminergic alternative, not only as symptomatic treatment, but also as potential neuroprotective therapy. Adenosine A2A receptors are co-localised with dopamine D2 receptors in the striatum and other nuclei of the basal ganglia. Adenosine A2A stimulation decreases the affinity of dopamine for the D2 receptor, and increase cyclic AMP (cAMP) levels. The stimulation of dopamine D2 receptors, in contrast, decreases cAMP levels and therefore these receptors (A2A and D2), act in an opposing manner. Adenosine A2A antagonism will thus have similar effects as dopamine D2 agonism and will reduce the postsynaptic effects of dopamine depletion to give symptomatic relief. There are also several mechanisms where by adenosine A2A antagonists may be neuroprotective, for example by preventing glutamate excitotoxicity, that may cause damage to dopaminergic neurons. A number of adenosine A2A antagonists have already reached clinical trials and promising results were obtained, especially when combined with levodopa. Consequently, A2A antagonists are realistic prospects that have therapeutic potential in diseases with dopaminergic hypofunction, like Parkinson's disease. Many of the current A2A antagonists contain an amino-substituted heterocyclic scaffold, such as an aminopyrimidine. The primary aim of this study was the design, synthesis and evaluation of 2-aminopyrimidine derivatives as adenosine A2A receptor antagonists. Monoamine oxidase B (MAO-B) inhibitors are also promising candidates for the symptomatic treatment of Parkinson's disease, since MAO-B is the enzyme primarily responsible for the catabolism of dopamine in the brain. Irreversible inhibitors of MAO-B, such as selegeline and rasagiline, have been used clinically for the treatment of Parkinson's disease. This type of inhibition comes with certain disadvantages as it may take up to several weeks after termination of treatment for the enzyme activity to recover. Reversible inhibitors in contrast will have much better safety profiles seeing that they will not inactivate the enzyme permanently and allow for competition with the substrate. When dopamine is oxidized by MAO, toxic metabolic by-products, such as hydrogen peroxide (H2O2) forms, and this is believed to be a possible cause of Parkinson's disease. MAO-B inhibitors will therefore not only provide symptomatic relief but may also alter the progression of the disease by preventing the formation of these byproducts. Promising MAOB inhibitory activities have been reported for chalcones, and since the intermediates obtained in the synthesis of aminopyrimidines in this study are chalcones, a secondary aim of this study was the screening of selected chalcone intermediates as inhibitors of MAO–B. Results - Design and synthesis: A series of 2-aminopyrimidines were designed using known active structures and literature pharmacophores. A molecular modelling study (Discovery Studio 3.1, Accelrys) was further done to investigate the feasibility of these compounds as potential adenosine A2A antagonists. All of the designed aminopyrimidines were successfully docked in the binding site of the adenosine A2A receptor. Binding orientations and observed interactions with important residues in the active site were similar to those observed for known A2A antagonists. It was therefore concluded that these compounds may be potential A2A antagonists and the designed compounds were thus synthesised. Structures were primarily confirmed with nuclear magnetic resonance spectroscopy and mass spectrometry. MAO-B inhibition studies: Selected chalcones were evaluated using a fluorometric assay and kynuramine as substrate. The compounds were potent and selective inhibitors of the MAO-B enzyme with IC50 values ranging between 0.49-7.67 μM. (2E)-3-(3-Chlorophenyl)-1- (5-methyl-2-furyl)prop-2-en-1-one (1c) was the most potent compound with an IC50 value of 0.49 μM and was approximately 60 times more selective towards MAO-B than MAO-A. Some preliminary structure activity relationships were derived, for example, phenyl substitution with an electron withdrawing chlorine group generally resulted in better activity than substitution with electron donating methoxy groups. Further investigation of structure activity relationships are however required as a very small series of chalcones were screened. Reversibility studies and mode of inhibition: A dilution assay was used to determine whether compound (1c) binds reversibly or irreversibly to the MAO-B enzyme. This was done by measuring the recovery of enzymatic activity after a large dilution of the enzyme-inhibitor complex. The results from the reversibility studies showed that the inhibition of the most potent compound (1c) is reversible as the catalytic activities are recovered to approximately 80% and 50% respectively, compared to the control measured in the absence of an inhibitor. For the mode of inhibition, sets of Lineweaver–Burk plots were constructed. The Lineweaver- Burk plots intersected on the y-axis which indicates that compound 1c is a competitive inhibitor of the MAO-B enzyme. In vitro adenosine A2A assays: Radioligand binding assays were used to determine the affinity of the synthesised 2-aminopyrimidines for the adenosine A2A receptor. This assay was performed with the radioligand [3H]NECA in the presence of N6-cyclopentyladenosine (CPA). Compounds 2a - 2h showed moderate to weak affinity in the assay, while promising affinities were observed for compounds 2j - 2n, which all exhibited Ki values below 55 nM. The compound with the highest affinity was 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine (2m) with a Ki value of 5.76 nM, which is comparable to the Ki value of 2.10 nM obtained for the known amino-substituted heterocyclic adenosine A2A antagonist, ZM 241385. The higher affinities of compounds (2j – 2n) could, at least in part, be explained by the molecular modellling studies. In the docking experiments an additional hydrogen bond interaction was observed between the amide carbonyl and tyrosine 271 indicating that this structural feature is a major contributing factor to the improved affinity observed for these derivatives. In vivo adenosine A2A assays: The haloperidol induced catalepsy assay was used to determine whether the two compounds with the highest affinity for the adenosine A2A receptor (2m and 2k) are antagonists of the A2A receptor. These compounds caused a statistically significant reduction in catalepsy, which clearly illustrate that they are adenosine A2A antagonists. The objectives of this study as set out were thus successfully realised and promising results were obtained. During this study, several novel 2-aminopyrimidines and chalcones were synthesised, and the respective adenosine A2A antagonistic and monoamine oxidase inhibitory activities for all of the screened compounds were determined for the first time.
Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

FOSCHETTI, D. A. Toxinas A e B do Clostridium difficille induzem a expressão diferencial de receptor de Adenosina em células epiteliais intestinais: papel do receptor A2B. 2014. 160 f. Tese(Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2014.
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Clostridium difficile is recognized to be a nosocomial pathogen that causes intense intestinal inflammation, epithelial barrier disruption and diarrhea. Adenosine production is increased under inflammatory situations. The adenosine receptor A2B is the most expressed receptor in the human and mice intestine. We investigated the effect of short- and long-term exposure to TcdA and TcdB in HCT-8 cells and isolated cecum epithelial cells. HCT-8 cells were exposed to TcdA or TcdB (10 ng/ml) for 2, 6 and 24h. We used a murine cecal loop model and murine infection model to evaluate the effects of TcdA and C. difficile infection, respectively. We demonstrated that HCT-8 and isolated intestinal cecum epithelial cells naturally express high levels of A2BR receptors. TcdA or TcdB alters the cell morphology, viability and proliferation pattern and caused gene expression increase of all AR subtypes in HCT-8. In isolated cecum epithelial cells, TcdA significantly (p<0.05) increased volume/length, weight/length, histopathology scores, neutrophil infiltration, as measured by MPO content, and induced an altered gene expression increase of all AR subtypes. PSB603 (10 nM) treatment significantly (p<0.05) reduced TcdA-induced tissue damage. Our findings support the hypothesis that Clostridium difficile toxins affect adenosine receptor expression and this action may be related to their severe inflammatory effect. We concluded that adenosine receptors may play a crucial role in regulating the inflammatory system in intestinal epithelium during C. difficile infection.
O Clostridium difficile é reconhecido por ser uma bactéria nosocomial, levando a intensa inflamação intestinal, quebra da barreira epitelial e diarreia. A produção de adenosina está aumenta durante eventos inflamatórios. O receptor de adenosina A2B (A2BR) é o mais expresso no intestino de humanos e camundongos. Nós investigamos o efeito de exposição às TcdA e TcdB, a curto e longo prazo, em células HCT-8 e células epiteliais intestinais isoladas do ceco. Células HCT-8 foram expostas a TcdA ou TcdB (10 ng/ml) por 2, 6 e 24h. Foi usado o modelo de alça cecal e de infecção pelo bacilo em murinos para avaliar os efeitos da TcdA e da infecção pelo C. difficile, respectivamente. Foi demonstrado que HCT-8 e células epiteliais intestinais isoladas do ceco naturalmente expressam altos níveis do receptor A2BR. TcdA e TcdB alteraram a morfologia celular, viabilidade e proliferação e causaram aumento da expressão gênica de todos os subtipos de receptores de adenosina e das citocinas IL-6 e IL-8 em HCT-8. Em células epiteliais intestinais isoladas do ceco, a TcdA significativamente causou um aumento do peso e volume/comprimento da alça cecal, escores histológicos e infiltrado neutrofílico, medido por MPO, e também causou alterações da expressão gênicas dos receptores de adenosina, tanto no modelo de alça cecal quanto na infecção pelo bacilo. O tratamento com PSB603, um antagonista do receptor A2BR, foi capaz de reverter os efeitos causados pelas toxinas do C. difficile. Nossos dados suportam a hipótese que as toxinas do C. difficile alteram a expressão dos receptores de adenosina e isso pode estar relacionado com os severos efeitos inflamatórios. Nós concluimos que os receptores de adenosina tenham um papel importante na regulação da inflamação em células epiteliais intestinais na infecção pelo C. difficile.

GPR37 is an orphan GPCR that belongs to the Rodhopsin family. GPR37 is highly expressed in the Central Nervous System (CNS), while little peripheral expression has been found in humans and rodents. GPR37 is highly distributed in the brain, particularly at the cerebellum, corpus callosum, substantia nigra, striatum and hippocampus with a predominantly neuronal distribution. However, the function of this receptor in the CNS remains unknown. The interest on GPR37 biology was bolstered when it was described as a Parkin substrate. GPR37 was isolated from a human brain library in a yeast 2-hybrid screen for novel interacting partners of Parkin, and thus rebaptized as “Parkin-associated endothelin-like receptor” (Pael-R). Parkin is of great interest because mutations in this gene are directly linked to autosomal recessive juvenile Parkinsonism (AR-JP). It was found that insoluble GPR37 was increased in patients with AR-JP, linking the aggregation of GPR37 to disease pathogenesis. Later, GPR37 aggregates were also found in a variety of inclusion bodies in brains from patients with PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). All these data suggested a relevant role of the GPR37 in the aggregates formation in the PD. Our working hypothesis is based on that this GPR37 oligomerization with the adenosine and dopamine receptors, together with the neuropathological changes during PD can be relevant during PD development and basal ganglia function, and thus for the PD ethiopatology. Finally, in addition to elucidate GPR37 function in the CNS, in this doctoral thesis we have studied different specific ligand candidates for this orphan receptor. RESULTS Elucidating the existence and functional consequences of a direct GPR37-A2AR receptor- receptor interaction in the striatum. Two polyclonal antibodies which recognized the extracellular N-terminal or the intracellular C-terminal fragment of GPR37 respectively were produced by the immunization of rabbits (strain New Zealand White) in the Animal house facilities in the Bellvitge Health Sciences Campus. The results obtained by Western Blot using the antibody against the N-terminal region of the GPR37 receptor was a single specific band of about 53 kDa, similar to the theoretical weight calculated for the receptor peptidic chain. On the other hand, the result obtained by Western Blot using the antibody against the C-terminal region of the GPR37 receptor was a single specific band of about 40 kDa, an unexpected result since the expected size of the complete GPR37 receptor is approximately 53 kDa. Our results confirmed this N-terminal cleavage of the receptor in native tissue. In our work, we studied with special emphasis the physical and functional interaction between the GPR37 and A2AR receptor in native tissue, specifically in striatum. First, we verified the presence, co-distribution and direct interaction of GPR37 and A2AR by different methods: i) Through histoblot and immunohistochemistry (IHC) we confirmed that the two receptors co-distribute in the striatum. ii) Next, by means of electron microscopy and the technique of subsynaptic fractionation we characterized the subsynaptic distribution of these receptors in the striatum, observing that both receptors were enriched at the postsynaptic level. iii) Using co-immunoprecipitation (Co-ip), the proximity ligation assay (PLA) technique and double labeling with gold immunoparticles (in collaboration with Prof. Rafael Luján, UCLM, Albacete), we demonstrated the direct interaction between both receptors in the striatum. Subsequently, we analyzed the impact of the presence/ absence of GPR37 on the expression and functionality of A2AR. Interestingly, GPR37 deletion promoted an increase in A2AR membrane expression in the striatum observed by biotinylation of membrane proteins performed on corticostriatal slices. On the other hand, we determined the generation of A2AR-mediated cAMP after stimulation with CGS21680, a selective agonist of this receptor, in total synaptosomes and primary cultures of striatum. Interestingly, the results showed a greater effect of the A2AR agonist on GPR37-/- mice when compared with the Gpr37+/+ mice. In addition, we performed behavioral tests to analyze striatal-dependent functions (i.e. locomotor activity) to assess the relevance of GPR37 receptor and its impact on A2AR function by administering SCH58261 (3.75mg/kg, intraperitoneal, i.p.), a selective A2AR receptor, in GPR37+/+ and GPR37-/- mice. In the Open field test, mice freely explored the field. The horizontal locomotor activity (total distance traveled) was analyzed and a greater increase of the spontaneous locomotor activity in the GPR37-/- animals treated with SCH58261 was observed. Finally, we characterize the role of GPR37 in an animal model of PD, and its relation with the adenosinergic transmission. PD is characterized by a degeneration of the nigrostriatal dopaminergic neurons, resulting in an imbalance of the function of the basal ganglia. Pharmacological treatment of PD is mainly based on improving dopaminergic transmission (either by enhancing the release of dopamine or by directly activating dopamine receptors). However, severe side effects appear after prolonged treatment, including dyskinesias and somnolence. During the last decades, the A2AR antagonists have been studied as a good alternative or supplementation (together with the classical dopaminergic drugs) for the treatment of PD. A2AR antagonists mechanism, is based on the interaction between these receptors and the D2R in the striatum. For example, A2AR antagonists attenuate catalepsy induced by D2R blockade in rodents. In addition, this catalytic effect can also be induced by the administration of A2AR agonists at high doses. We used the pharmacological catalepsy model to evaluate the impact of the presence of GPR37 on the adenosinergic transmission in the striatum. First, we administered haloperidol, a D2R antagonist, together with SCH58261, and quantified the duration of the cataleptic effect. Next, we used the CGS21680, a selective A2AR agonist, administered intracerebroventricularly (i.c.v.), to cause the cataleptic effect. The results showed an increase in the cataleptic response in GPR37-/- animals which would correlate with an increase in the A2AR expression in the synapse previously observed. In order to study the role of this orphan receptor in CNS synaptic plasticity and to evaluate its involvement in A2AR receptor function, we performed behavioral experiments related to striatal functions in GPR37+/+ and GPR37-/- mice of 2 months old mice administered chronically with SCH58261 (1mg/kg, i.p. for 14 days). Thus, we performed the following behavioral tests: i) Open field test and ii) Modified Water maze test. These tests allowed us to evaluate locomotor activity and learning memory, respectively. Subsequently, extracellular field electrophysiological recordings were performed on corticostriatal slices to evaluate possible changes in the synaptic activity and plasticity of these animals, and their relationship with GPR37 receptor or chronic treatment with the A2AR antagonist. In conclusion, the results obtained in the behavioral experiments and LTD electrophysiological records performed in corticostriatal slices, made us hypothesize that in the absence of the GPR37 receptor, sensitization to A2AR receptor antagonists occurs, due to differences in the levels of this receptor in the membrane. This would coincide with our results previously obtained in the laboratory, where using techniques such as biotinylation of membrane receptors or subsynaptic fractionation, where we can quantify the amount of receptors in pre- and postsynaptic densities, we observed an increase in A2AR receptor expression in the plasma membrane in the Gpr37 - / - animals. Establishing the existence of GPR37-A2AR in other brain areas, such as the hippocampus, describing the synaptic distribution of GPR37 and determining its role in A2AR-mediated synaptic plasticity. The results obtained with this work described the presence of this orphan receptor in the hippocampus and a preferably postsynaptic location in the neurons. In addition, a series of behavioral tests related to hippocampal dependent functions (i.e. anxiety and memory) were also performed to evaluate the relevance of the GPR37. We also evaluated the interaction of GPR37 with the A2AR by administering SCH58261. Novel Object Recognition (NOR) test, widely used to study effects on memory. On the other hand the Marble Burying Test (MBT) and the Elevated Plus Maze (EPM) were performed to evaluate the anxiety. The results obtained coincide with previous studies, which showed that the deletion of A2AR has an anxiogenic effect and that overexpression leaded to a less anxious phenotype. Evaluating GPR37 receptor expression levels in PD and the potential use of this receptor as a PD biomarker. The expression/ recycling of GPR37 in some types of PD is altered, triggering an increase and accumulation of this receptor in neurons. In this objective, we intend to describe whether this process occurs only in an isolated group of PD patients (AR-JP, a genetic cause) or if it was a more widespread process in PD, thus being able to describe its histopathological evolution through the different stages of the disease. In addition, this increase of the GPR37 receptor in the tissues of the CNS could translate into an increase in the presence of the receptor or fragments of this receptor in body fluids such as blood or cerebrospinal fluid (CSF). This process is known for other proteins such as α-synuclein, Phospho-Tau, β-amyloid, related CNS diseases. At this moment we are currently completing the validation of a system for the detection of the ecto-GPR37 receptor (a N-terminal fragment of the GPR37 produced and liberated after metalloproteinase cleavage) in CSF which could be used as a new biomarker for earlier and finer diagnosis in PD.
INTRODUCCIÓN GPR37 es un receptor huérfano acoplado a proteína G que se encuentra casi exclusivamente en el sistema nervioso central (SNC), enriquecido en áreas tales como el cerebelo, cuerpo calloso, bulbo raquídeo, putamen, núcleo caudado, sustancia negra e hipocampo. Sin embargo, la función de este receptor en el SNC es aún desconocida. El interés por GPR37 se basa en su identificación, hace unos pocos años, como sustrato de Parkin. Parkin es una ubiquitina ligasa E3 proteasomal que participa en la ubiquitinación y la degradación/eliminación de proteínas mal plegadas. Esta pérdida de función es característica de la enfermedad de Parkinson Juvenil (AR- JP), en la que se produce una mutación en Parkin y consecuentemente una pérdida parcial de su función. Tal pérdida conduce a una acumulación tóxica de los sustratos de Parkin (como GPR37). Además, se ha descrito la sobreexpresión de GPR37 en cerebros de pacientes de AR- JP, así como su presencia en el núcleo de los cuerpos de Lewy de pacientes de la Enfermedad de Parkinson (EP). Estos hechos sugieren un papel importante de los agregados de GPR37 en la EP. Nuestra hipótesis de trabajo se basa en que la oligomerización del receptor GPR37 con receptores de adenosina y dopamina, así como los cambios que se producen durante la enfermedad, es relevante para la función de los ganglios basales y por tanto en la etiopatología de la EP. Finalmente, cabe añadir que además de elucidar la posible función de GPR37 en el SNC, en la presente tesis doctoral hemos intentado también encontrar un ligando específico de este receptor huérfano. RESULTADOS Establecer el papel del GPR37 en el estriado y su impacto sobre la función del A2AR. En esta tesis doctoral, se han desarrollado en el laboratorio dos nuevas herramientas para la detección y estudio del receptor huérfano GPR37: un anticuerpo policlonal producido en conejo (cepa New Zealand White) que reconoce el fragmento N-terminal y otro anticuerpo que reconoce el fragmento C-terminal de este receptor. El resultado obtenido en el Western Blot usando el anticuerpo contra la región N-terminal del receptor GPR37 fue una única banda específica de aproximadamente unos 53 kDa, similar al peso teórico calculado para la cadena peptídica del receptor. Por otro lado, el resultado obtenido en el Western Blot usando el anticuerpo contra la región C-terminal del receptor GPR37 fue una única banda específica de aproximadamente unos 40 kDa, hecho sorprendente ya que el tamaño esperado del receptor completo GPR37 es de aproximadamente 53 kDa. En paralelo a este hallazgo, fue publicado un artículo en el que se describía la acción de unas metaloproteasas sobre este receptor en un sistema de expresión heterólogo, cortando al receptor GPR37 en su dominio N-terminal extracelular. Nuestros resultados confirman este hecho, pero además incrementan la discusión sobre dónde actúan estas metaloproteasas y de si tienen solo un punto o varios de corte en el dominio extracelular del receptor ya que la diferencia entre el receptor entero y el cortado sería de unos 20 kDa. Además, el hecho de detectar una única banda de unos 40 kDa con el anticuerpo GPR37 que reconoce el epítopo intracelular indica que el receptor se encuentra principalmente cortado. En nuestro trabajo, hemos estudiado con especial énfasis la interacción física y funcional entre el receptor GPR37 y A2AR en tejido nativo, concretamente en estriado. En primer lugar, comprobamos la presencia, co-distribución e interacción directa del GPR37 y A2AR por diferentes métodos: i) Mediante histoblot e inmuohistoquímica (IHC) confirmamos que los dos receptores co-distribuyen en el estriado. ii) Mediante microscopia electrónica y la técnica de fraccionamiento subsináptico determinamos la distribución subsináptica de estos receptores en el estriado, observando que ambos receptores están enriquecidos a nivel postsináptico. iii) Mediante co-immunoprecipitación, la técnica proximity ligation assay (PLA) y doble marcaje con inmunopartículas de oro para la observación mediante microscopia electrónica, en colaboración con el Prof. Rafael Luján (UCLM, Albacete), demostramos la interacción directa entre ambos receptores en el estriado. Posteriormente, hemos analizado el impacto de la presencia/ausencia de GPR37 en la expresión y funcionalidad del A2AR. De forma interesante, mediante biotinilización de las proteinas de membrana realizado en rodajas corticoestriatales, se observó un incremento de la expresión en membrana del A2AR en ausencia de GPR37 en el estriado. Por otro lado, determinamos la generación de AMPc mediada por A2AR tras la estimulación con CGS21680, un agonista selectivo de este receptor en sinaptosomas totales y cultivos primarios de estriado. De forma interesante, los resultados muestran un mayor efecto del agonista A2AR en los animales GPR37-/- respecto a los GPR37+/+, reforzando nuestros resultados previos obtenidos en células HEK293T (no se muestran los datos). Además, también se realizaron tests de conducta relacionados con funciones dependientes del estriado (actividad locomotora) para evaluar la relevancia del receptor GPR37 y su impacto sobre la función del A2AR administrando SCH58261 (3.75mg/kg, intraperitoneal, i.p.), un antagonista selectivo del receptor A2AR, en ratones GPR37+/+ y GPR37-/-. El test empleado fue el test de campo abierto, que consiste en exponer a los animales, durante un periodo de 10 minutos en condiciones de luz tenue a un campo de 30x30cm. Durante este periodo, los ratones exploran libremente el campo. La actividad locomotora horizontal (distancia total recorrida) fue analizada observándose un mayor aumento de la actividad locomotora espontanea en los animales GPR37- /-. Finalmente, caracterizamos la función del GPR37 en un modelo de la EP y su relación con la transmisión adenosinérgica. La EP está caracterizada por una degeneración de las neuronas dopaminérgicas nigroestriatales, que resulta en un desequilibro de la función de los ganglios basales. El tratamiento farmacológico de la EP se basa principalmente en la mejora de la transmisión dopaminérgica (ya sea potenciando la liberación de dopamina o activando directamente los receptores de dopamina). Sin embargo, efectos secundarios severos aparecen tras el tratamiento prolongado, entre los cuales se incluyen disquinesias y somnolencia. Los antagonistas A2AR se han estudiado desde ya hace un tiempo como una buena alternativa/complementación para el tratamiento de la EP. Su empleo se basa en la interacción entre estos receptores y los D2R en el estriado. Por ejemplo, los antagonistas del A2AR atenúan la catalepsia inducida por el bloqueo de D2R en roedores. Además, este efecto cataléptico también puede inducirse mediante la administración de agonistas del A2AR a dosis elevadas. Utilizamos el modelo de catalepsia inducida farmacológicamente para evaluar el impacto de la presencia del GPR37 en la transmisión adenosinérgica en el estriado. Primero, administramos haloperidol, un antagonista D2R, juntamente con SCH58261, antagonista A2AR, y se cuantificó la duración del efecto cataléptico. Los resultados obtenidos no fueron suficientemente claros debido a que el ratón GPR37-/- tiene alterada la transmisión dopaminérgica, así decidimos usar el CGS21680, agonista A2AR, administrado intracerebroventricularmente (i.c.v.), para causar el efecto cataléptico. Los resultados muestran un incremento en la respuesta cataléptica en los animales GPR37-/-. Este hecho coincide con los resultados obtenidos por fraccionamiento subsináptico, en los que también se observó un incremento en la expresión del A2AR en la sinapsis. Con el fin de estudiar la función del receptor huérfano GPR37 en la plasticidad sináptica del SNC, y evaluar su implicación en la función del receptor A2AR, realizamos experimentos conductuales relacionados con funciones estriatales en ratones GPR37+/+ y GPR37-/- de 2 meses de edad administrados con SCH58261 (1mg/kg, i.p.) durante 14 días. La dosis del antagonista A2AR, fue elegida en base a experimentos previos realizados en nuestro grupo, donde se observó que una administración aguda de este fármaco a esta concentración, no producía una alteración de la actividad locomotora. Así, realizamos las siguientes pruebas conductuales: i) Test de campo abierto y ii) Test de Water maze modificado. Estos test nos permitieron evaluar la actividad y el aprendizaje locomotor, respectivamente. Posteriormente, se realizaron experimentos de electrofisiología en rodajas corticoestriatales para evaluar posibles cambios en la actividad y plasticidad sinápticas de estos animales, y su relación con receptor GPR37 o al tratamiento crónico con el antagonista A2AR. En conclusión, los resultados obtenidos en los experimentos de conducta y registros electrofisiológicos de LTD realizados en rodajas corticoestriatales, nos hacen hipotetizar que en ausencia del receptor GPR37, ocurre una sensibilización a antagonistas del receptor A2AR, debido a diferencias en los niveles de este receptor en la membrana. Esto coincidiría con nuestros resultados obtenidos previamente en el laboratorio, donde usando técnicas como biotinilización de receptores de membrana o fraccionamientos subsinápticos, donde podemos cuantificar la cantidad de receptores en las densidades pre- y postsinápticas, hemos observado un incremento en la expresión del receptor A2AR en la membrana plasmática en los animales Gpr37-/-. Caracterizar la función del GPR37 en el hipocampo y su relación con la transmisión adenosinérgica. Con la finalidad de estudiar la presencia y localización del receptor GPR37 en el hipocampo, realizamos cultivos primarios y fraccionamiento subsináptico de hipocampo para determinar la localización de este receptor en las neuronas, donde se observó la presencia de este receptor huérfano en el hipocampo y una localización preferentemente postsináptica en las neuronas. Además, también se realizaron una serie de tests de conducta relacionados con funciones dependientes del hipocampo (ansiedad y memoria) para evaluar la relevancia del receptor GPR37. Igualmente, evaluamos la interacción de GPR37 con el A2AR administrando SCH58261, un antagonista selectivo de este último receptor. El primer test empleado fue el test de Novel Object Recognition (NOR), ampliamente utilizado para estudiar efectos sobre la memoria. Este test consta de dos sesiones, en la primera se le presentan dos objetos iguales al ratón (fase de familiarización) y dos horas después se realiza otra sesión presentándole un objeto nuevo (fase de evaluación). Basándose en el tiempo de exploración de cada objeto (objeto familiar vs nuevo) se calcula el % de preferencia por el objeto nuevo (tiempo de exploración del objeto nuevo/tiempo de exploración total). En el NOR no se observaron diferencias entre los animales GPR37+/+ y GPR37-/- en la retención de memoria pero sí una potenciación del efecto de SCH58261. Para evaluar otras características emocionales como la ansiedad realizamos el Marble Buring Test (MBT) y el Elevated Plus Maze (EPM). El MBT consiste en evaluar una conducta innata en los animales como es el excavar/enterrar ya sea como una respuesta defensiva ante un objetivo que les produce miedo o para proteger la comida de otros depredadores. En este test se le presentan al ratón 9 canicas colocadas equidistantemente y a los 30 minutos se contabilizan el número de enterradas. Los resultados obtenidos muestran que los ratones GPR37+/+ enterraron más canicas que los GPR37-/-, pudiéndose interpretar como un fenotipo menos ansioso en los animales GPR37-/-. Además al administrar el SCH este fenotipo se revirtió, confirmando la interacción de GPR37 con el A2AR. Los resultados obtenidos coinciden con estudios realizados anteriormente, en los que se demostró que la deleción del A2AR tiene un efecto ansiogénico que la sobreexpresión conduce a un fenotipo menos ansioso. Finalmente, el test más ampliamente utilizado para evaluar ansiedad es el EPM. El EPM consiste en colocar al ratón durante 10 minutos en un aparato en forma de cruz con dos de sus brazos abiertos y dos cerrados, elevado a 40 centímetros del suelo y se contabiliza el tiempo explorando los brazos abiertos. Mediante este test se evalúa la actividad exploratoria de los ratones en un instrumento con dos ambientes, uno más estresante que otro ya que los brazos cerrados ofrecen un entorno menos estresante y expuesto que los abiertos. De esta manera una mayor exploración de los brazos abiertos indica un estado menos ansioso del animal. En el EPM se observó una mayor exploración de los brazos abiertos en los animales GPR37-/- que en los GPR37+/+. Además, al administrar el antagonista del A2AR se reprodujo un efecto ansiogénico del fármaco en los animales GPR37-/-. Caracterizar los niveles de GPR37 en diferentes etapas de la enfermedad de Parkinson. Como ya se ha dicho con anterioridad, la expresión/reciclaje del GPR37 en algunos tipos de EP se encuentra alterada, desencadenando un incremento y acúmulo de este receptor en las neuronas. En este objetivo pretendemos describir si este proceso sólo ocurre de manera aislada en la AR-JP (causa genética) o si se trata de un proceso más extendido en la EP de origen idiopático, pudiendo así describir su evolución histopatológica en las diferentes etapas de la enfermedad. Además, este incremento del receptor GPR37 en los tejidos del SNC podría traducirse en un incremento en la presencia del mismo, ya sea en su totalidad o fragmentados de este receptor, en fluidos del cuerpo como la sangre o el líquido cefaloraquídeo (CSF). Este proceso es conocido para otras proteínas como la α-sinucleina, Phospho-Tau, β-amyloid, relacionadas con enfermedades del SNC. Actualmente, estamos acabando la validación de un sistema para la detección del receptor GPR37 en CSF. Si éste fuera el caso, podría usarse como un nuevo biomarcador para un diagnóstico precoz y más fino en la EP.

The aim of this work was the design and synthesis of new A2A and A3 adenosine receptors antagonists. Two different studies have been performed. The first one based on structural modifications of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pirimidine nucleus, reported in literature. In order to identify a new series of A2A or A3 AR antagonists and with the aim to better investigate the role of the nitrogen at the 7- position on the interaction with ARs, it was performed a synthetic strategy for the preparation of the pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus which can be considered the 7-deaza-analogue of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine core. In order to complete the SAR studies on this class of compounds, it has been synthesised a novel series of pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives which can be considered the structural isomers of the parent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. From the biological data obtained, we can assert that the concomitant presence of the N7 and N8 is fundamental for the selectivity of these A2A/A3 ligands versus the remaining ARs subtype. Our results confirmed the importance of the presence of the NH at the 5- position of the PTPs nuclus, this could be due to the formation of an essential ligand-receptor hydrogen bond. Most of these compounds, revealed to be non selective ARs antagonists. Derivative 5-{[(4-methoxy-phenyl)carbamoyl]amino}-(2-furan-2-yl)-8-methyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine resulted to be the best compound of the series in term of both affinity (hA3Ki = 15nM) and selectivity. The aim of the second project was to obtain A3 antagonists with improve water-solubility. A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/ethyl-2-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-one derivatives have been synthesised and evaluated in radioligand binding assays to determine their affinities at the human A1, A2A, and A3 adenosine receptors. Efficacy at the hA2B AR and antagonism of selected ligands at the hA3 AR were also assessed through cAMP experiments. All the synthesised molecules exhibited high affinity at the hA3 AR (Ki values ranging from 1.46 to 44.8 nM) as well as remarkable selectivity versus A1, A2A and A2B AR subtypes. In view of the chirality of the tricycles, for selected compounds both the racemic mixtures and the pure enantiomers have been prepared with the purpose of determine a possible receptor stereoselectivity. Compound (R)-4-allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one showed to be the most potent hA3 AR ligand of the series (hA3Ki = 1.46 nM), in addition with very high selectivity over all the other ARs (hA2AKi/ hA3Ki > 3425; hA2BIC50/ hA3Ki > 3425; hA1Ki/ hA3Ki = 1,729).

The difficulty with movement initiation, or akinesia, is a cardinal symptom of Parkinson�s disease (PD) and the loss of dopaminergic cells, affecting the function of the basal ganglia, the thalamus and the motor cortex, has long been documented. From a broader perspective, it has been proposed that akinesia is caused by impaired function in different brain areas, inside and outside the basal ganglia, operating as a �behavioural arrest control system� (Klemm, 2001). Several neurotransmitters seem to modulate the activity of this system and, contrasting the well-known effects of dopamine, the involvement of adenosine has only recently emerged, particularly via A2A receptors. Adenosine plays an opposite role to dopamine in the brain: adenosine stimulation at A2A receptors inhibits movement (Ferre et al., 1991a; Hauber and Munkle, 1995; Rimondini et al., 1997), whereas A2A antagonists seem to promote movement (Kanda et al., 2000; Bara-Jimenez et al., 2003; Pinna et al., 2005). Although specific adenosine A2A and dopamine D2 receptors are known to antagonistically interact (Ferre et al., 1997; Fuxe et al., 1998; Ferre et al., 2001), little is known of the involvement of A2A receptors in regulating neural activity in the basal ganglia, a crucial point for the future use of A2A antagonists as adjuvant therapy in Parkinson�s disease. In fact, although it is generally accepted that akinesia results from altered function in the cortico-basal ganglia-cortical loop, as confirmed in several studies reporting changes in basal ganglia activity following dopamine depletion (Blandini et al., 2000; Bevan et al., 2002; Boraud et al., 2002), no study to date has systematically investigated electrophysiological changes in the basal ganglia during akinesia induced by adenosine receptor stimulation. Starting from a common behavioural effect, this study tries to bridge this gap by investigating and comparing, in two basal ganglia structures, the neural substrate of akinesia after acute dopamine D2 receptor blockade and adenosine A2A receptor stimulation. The external segment of the globus pallidus (GP, or simply globus pallidus in the rat) and the substantia nigra pars reticulata (SNr) were chosen as the recording sites because both nuclei are included into the �behavioural arrest control system� and seem to express somewhat complementary functions, as a respective key integrative station and main output of the basal ganglia. Dopamine function was manipulated by acute decrease in availability of dopamine binding sites in the brain, through specific dopamine D2 receptor blockade with systemic injections (1.0 and 1.5 mg/kg) of raclopride(3,5-dichloro-N-[(1-ethylpyrrolidin-2-y)methyl]-2-hydroxy-6-methoxy-benzamide), resulting in akinesia. Conversely, movement was inhibited by specific adenosine A2A receptor stimulation with systemic injections (2.5 and 5.0 mg/kg) of the drug CGS21680 (sodium-2-p-carboxyethylphenylamino-5-N-carboxamidoadenosine). In both situations, behaviour was assessed through specific akinesia tests. Single neuron activity before injection and changes in the firing frequency and firing pattern occurring after injection have been analysed and compared for each cell recorded from GP and SNr, during periods of behavioural rest. Synchronised firing between cell pairs has also been assessed. However, the small number of cell pairs showing correlated firing in each structure after systemic injection of drugs was not statistically relevant for further analysis and interpretation of synchronised firing during drug induced akinesia. In our experiments, both drugs inhibited movement, albeit somewhat differently, with lack of rigidity and �flat� body position after adenosine stimulation. Dopamine blockade decreased mean firing rate and dramatically altered the firing pattern in both investigated structures, generally increasing burst activity (increased percentage of spikes in bursts, mean number of bursts, mean number of spikes per burst, mean intra-burst firing frequency) and decreasing regularity of firing (increased coefficient of variation of the inter-spike intervals). Increased burst activity in the rat basal ganglia in an acute model of parkinsonian akinesia, following systemic raclopride injections, confirmed the importance of changes in the firing pattern in PD. The only electrophysiological effect of systemic A2A stimulation was decreased mean firing rate in the GP, a weak effect that could not propagate towards output stations of the basal ganglia. The lack of changes in the firing pattern, at both input and output levels of the basal ganglia, suggests a correlation with the lack of rigidity in adenosine-stimulation-induced akinesia.

A transmissão sináptica é essencial para o funcionamento do sistema nervoso. A neuromodulação permite regular esse processo de forma precisa. Um desses mecanismos modulatórios é a regulação da liberação de neurotransmissores. A adenosina é um importante modulador da transmissão sináptica. Além disso, a ativação do subtipo A2a dos receptores para adenosina está envolvida com a facilitação da liberação de neurotransmissores no sistema nervoso central. O presente trabalho teve como objetivo avaliar os efeitos modulatórios da ativação do receptor A2a de adenosina sobre a liberação de neurotransmissores e sua via de sinalização intracelular em modelo in vitro. Além disso, a tese contempla a construção histórica dos conceitos abordados no trabalho permitindo uma visão clara de sua evolução. Esse projeto foi o pioneiro no Brasil a utilizar o sensor biossintético fluorescente de liberação de vesículas sinápticas (supereclipse sinapto-pHluorina), o qual foi gentilmente cedido pelo professor Gero Miensenboeck do Sloan-Kettering Institute for Cancer Research. Nossos resultados demonstraram que o tratamento com o agonista do receptor A A2a de adenosina aumentou a fluorescência do supereclipse sinapto-pHluorina, assim como os níveis de glutamato e noradrenalina. Além disso, foi demonstrado que o inibidor da proteína cinase dependente de AMPc aboliu o aumento nos níveis do glutamato e noradrenalina, tal como a fosforilação da proteína sináptica sinapsina I evocado pelo agonista do receptor A2a de adenosina. Desta forma, nossos dados sugerem que a ativação do receptor A2a de adenosina em cultura de células do bulbo de ratos Wistar modula a liberação de neurotransmissores e a fosforilação da sinapsina I, assim como a proteína cinase dependente do AMPc pode ser o modus operandi desse fenômeno modulatório
Synaptic transmission is a sine qua non process for nervous system physiology. Such precise process is accomplished in part due to modulation of neurotransmitter release. Adenosine is a putative synaptic transmission modulator. Moreover, adenosine A2a receptor facilitates neurotransmitter release in the Central Nervous System. The present study focuses on the modulation of neurotransmission by adenosine A2a receptor and its intracellular signaling pathway in in vitro model. Here, we provided evidence that adenosine A2a receptor agonist increases an optical biosynthetic sensor of synaptic vesicle release (supereclipct synapto-pHluorin), as well as glutamate and noradrenaline. Furthermore, it was demonstrated that cAMP-dependent protein kinase inhibitor abolished glutamate and norepinephrin increase, as well as synapsin I phosphorylation evoked by adenosine A2a receptor agonist. Therefore, our data suggest that adenosine A2a receptor activation modulates neurotransmitter release and synapsin I phosphorylation in cultured cells from medulla oblongata of Wistar rats, as well as cAMP-dependent protein kinase might be the modus operandi of this modulatory phenomenon

O canabidiol (CBD), o principal canabinóide não psicotrópico extraído da marijuana (Cannabis sativa), é reconhecido por sua potente propriedade imunosupressora e anti-inflamatória. A injúria pulmonar aguda (ALI) é uma doença inflamatória para qual ainda não foi desenvolvida terapias específicas e a única alternativa de tratamento é meramente de suporte em UTI. Desta forma, foi proposta uma investigação sobre os efeitos anti-inflamatórios do CBD em um modelo murino de ALI, induzida pela instilação intra-nasal de LPS (lipopolissacarídeo), dentro de uma perspectiva imune-neuro-endocrinológica. Para a análise do potencial anti-inflamatório do CBD, avaliou-se a contagem total e diferencial de células do lavado broncoalveolar (LBA) (análise da migração de leucócitos para os pulmões), a atividade de mieloperoxidase (MPO) no tecido pulmonar (análise indireta da atividade de neutrófilos), a produção de citocinas e quimicionas no sobrenadante do LBA (análise do perfil inflamatório pulmonar), a concentração de proteínas (albumina) no sobrenadante do LBA (análise indireta da permeabilidade vascular pulmonar) e a expressão de moléculas de adesão (ICAM-1 e VLA-4) em leucócitos do LBA. Analisou-se, ainda, o mecanismo farmacológico dos efeitos anti-inflamatórios do CBD no modelo de ALI, utilizando-se de um antagonista altamente seletivo para o receptor de adenosina A2A (ZM241385). Por fim, avaliou-se os efeitos neuroendócrinos do CBD na vigência da inflamação pulmonar; analisou-se a atividade geral dos animais no campo aberto (análise do comportamento doentio) e os níveis séricos de corticosterona (análise da ativação do eixo Hipotálamo-Hipófise-Adrenal (HPA)). Mostrou-se que tanto o tratamento prolifático (antes da indução da inflamação) como o tratamento terapêutico (depois da indução da inflamação), com uma dose única de CBD (20 ou 80 mg/kg) apresenta um efeito anti-inflamatório prolongado em camundongos submetidos ao modelo de ALI (principalmente 1 e 2 dias após a indução da inflamação). Mostrou-se que o CBD diminuiu a migração de leucócitos para os pulmões (neutrófilos, macrófagos e linfócitos), diminuiu a produção de citocinas (TNF e IL-6) e quimicinas (MCP-1 e MIP-2) no LBA, diminuiu a atividade MPO no tecido pulmonar, diminuiu a concentração de albumina no LBA e diminuiu a expressão de moléculas de adesão (ICAM-1) em neutrófilos do LBA. Mostrou-se, ainda, que o receptor de adenosina A2A está envolvido nos efeitos anti-inflamatórios do CBD na ALI, uma vez que o tratamento com o ZM241385 aboliu todos os efeitos anti-inflamatórios descritos previamente. Por fim, mostrou-se que o CBD apresentou poucos efeitos comportamentais no campo aberto e que não ativou o eixo HPA. Desta forma, mostrou-se pela primeira vez que o tratamento profilático e, também, o tratamento terapêutico com CBD (20 ou 80 mg/kg) tem um efeito anti-inflamatório prolongado em um modelo murino de ALI, muito provavelmente em decorrência de um aumento da sinalização via receptor de adenosina A2A. Por esta razão, acredita-se que o CBD possa ser considerado, no futuro, uma ferramenta terapêutica útil no tratamento de doenças inflamatórias pulmonares.
Cannabidiol (CBD), the major non-psychotropic plant (Cannabis sativa)-derived cannabinoid, is recognized for its immunossupressant and anti-inflammatory properties. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive (ICU patients), because effective therapies have not been developed. Therefore, it was proposed an investigation in order to address the anti-inflammatory effects of CBD in a murine model of LPS-induced ALI, within an immune-neuro-endocrine perspective. To analyze the potential anti-inflammatory effect of CBD, it was evaluated total and differencial cell count of leukocytes present in the bronchoalveolar lavage (BAL) (migration of leukocytes into the lungs), myeloperoxidase activity in the lung tissue (indirect analysis of neutrophil activity), production of cytokines and chemokines in the BAL (analysis of the pulmonar inflammatory profile), protein (albumin) concentration in the BAL (indirect analysis of pulmonar vascular permeability), and expression of adhesion molecules (ICAM-1 and VLA-4) in leukocytes of the BAL. It was also analyzed the pharmacologic mechanism of the anti-inflammatory effects of CBD in the model of ALI, by using a highly selective antagonist of the adenosine A2A receptor (ZM241385). Finally, it was analyzed the neuro-endocrine effects of CBD in the context of lung inflammation; it was analyzed the general activity of the mice in the open field (analysis of sickness behavior) and the seric levels of corticosterone (activation of HPA (Hypothalamus-Hypophysis-Adrenal) axis). It was shown that both prophylactic (before the induction of inflammation) and therapeutic (after the induction of inflammation) protocols of treatment, with a sigle dose of CBD (20 or 80 mg/kg), has a long-term anti-inflammatory effect in mice submitted to the model of ALI (specially, after 1 and 2 days of the induction of inflammation). It was shown that CBD decreased leukocyte (neutrophil, macrophage, and lymphocytes) migration into the lungs, decreased cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) in the BAL, decreased MPO activity in the lung tissue, decreased albumin concentration in the BAL, and decreased adhesion molecule expression (ICAM-1) in neutrophils of the BAL. It was also shown that adenosine A2A receptor is involved in the anti-inflammatory effects of CBD on LPS-induced ALI, because ZM241385 abrogated all of the anti-inflammatory effects of cannabidiol previously described. Finally, it was shown that CBD has discreet behavioral effects in the open field and was not able to activate the HPA axis. Thus, it was shown for the first time that both prophylactic and also therapeutic treatment with CBD (20 or 80 mg/kg) has a long-term anti-inflammatory effect in a murine model of ALI, most likely associated with an increase in the signaling through the adenosine A2A receptor. Hence, it is possible that in the future CBD may prove useful as a therapeutical tool in the treatment of pulmonar inflammatory conditions.

Fibrose hepática é caracterizada pelo acúmulo de matriz extracelular fibrótica, cuja presença danifica as funções do fígado. Células estreladas hepáticas (HSCs) participam ativamente deste processo, modificando seu fenótipo quiescente, rico em lipídios no citoplasma, para o fenótipo ativado, em resposta ao insulto fibrogênico. A regressão do processo fibrótico pode, inclusive, estar relacionada com a apoptose das HSCs e também com sua reversão fenotípica, do estado ativado ao estado quiescente. Adenosina tem papel hepatoprotetor bem conhecido e medeia várias ações antiinflamatórias em diferentes tipos celulares e condições patológicas. TNF-α é uma citocina pró-inflamatória com importantes funções no início e perpetuação do processo fibrogênico. Tendo em vista o papel antiinflamatório da adenosina, este trabalho investigou, em linhagem de HSC em cultura - GRX -, as ações desse nucleosídeo em presença ou ausência dos sinais inflamatórios que acompanham o tratamento com TNF- α. Assim, foram analisados os efeitos da adenosina extracelular na síntese lipídica, avaliando a reversão fenotípica da GRX, e a apoptose em resposta à adenosina e/ou TNF-α. Além disso, a regulação dos níveis de adenosina extracelular, bem como a presença dos receptores de adenosina foram investigadas. O efeito de adenosina e/ou TNF-α sobre a produção de óxido nítrico (NO) e atividade e expressão de gelatinases (MMP-9 e -2), ambos importantes mediadores na fibrose hepática, também foram alvo deste estudo. Nossos resultados mostram a presença do receptor de adenosina A2B (A2BR), e a regulação dos níveis extracelulares de adenosina por TNF-α, através do aumento da atividade ecto-adenosina deaminase. Além disso, demontramos que adenosina extracelular não modifica a síntese de lipídios nas células GRX. Os dados ainda indicam que adenosina, em presença ou ausência de TNF-α, não resulta em apoptose, e que esta citocina também não induz à formação de corpos apoptóticos nas células tratadas. O estudo mostra que a produção de NO foi aumentada com TNF-α, com potencialização deste efeito na presença de adenosina, provavelmente mediado pelo A2BR e não por inosina, produto da hidrólise de adenosina. Com relação às gelatinases, o tratamento com adenosina diminui a atividade de MMP-9 tanto em ausência, quanto em presença de TNF-α, revertendo a ação da citocina, a níveis de controle, com o envolvimento do receptor A2BR mediando os efeitos do nucleosídeo. No entanto, a expressão da MMP-9 não foi afetada pelo tratamento com adenosina, porém, o nucleosídeo diminui os efeitos de TNF-α sobre a expressão da gelatinase. Com relação à MMP-2, ambos tratamentos com adenosina e com TNF-α, bem como o tratamento com adenosina, em presença da citocina, diminuem a atividade da gelatinase, sem efeitos aditivos. A expressão do mRNA de MMP-2 aumentou em resposta aos tratamentos com adenosina e TNF-α, isolados ou em associação, indicando a presença de mecanismo pós-transcricional de regulação para MMP-2. Este trabalho mostra, claramente, que adenosina extracelular e TNF-α estão envolvidos na regulação da produção de NO e nas ações das gelatinases. Além disso, este estudo demonstrou que adenosina não atua através de conversão fenotípica via aumento da síntese de lipídios, bem como não estimula apoptose, junto com TNF-α. Estes resultados e a existência da regulação dos níveis de adenosina extracelular por TNF-α sugerem uma participação da adenosina em alguns aspectos importantes da fisiologia da.HSC, talvez via A2B.
Hepatic fibrosis is characterized by fibrotic matrix accumulation, which damage the liver functions. Hepatic stellate cells (HSC) participate activelly of this process, modifying their quiescent phenotype, with the cytoplasm rich in lipid dropplets, to activated phenotype, in response to the fibrogenic insult. HSC can be responsible for the regression of fibrosis through mechanisms that involve their apoptosis, or even the phenotype reversion to quiscence. Adenosine has a well-know hepatoprotective role and mediates several anti-inflammatory actions in different cellular types and pathological conditions. TNF-α is a pro-inflammatory cytokine with important functions in the beginning and perpetuation of that fibrogenic process. In view the antiinflammatory role of adenosine, this work investigated, in cultured hepatic stellate cell line - GRX -, the actions of this nucleoside in the presence or absence of inflammatory signs that come with treatment with TNF-α. So, the effects of extracellular adenosine and/or TNF-α on the lipid synthesis, evaluating the phenotypic reversion of GRX, and the apoptosis in response to adenosine and/or TNF-α were analysed. Moreover, the regulation of extracellular levels of adenosine, as well as the presence of adenosine receptors were studied in cultured GRX. The effects of adenosine and/or TNF-α production of nitric oxide (NO) and activity and expression of gelatinases - MMP-9 and -2 -, both important mediators presents in liver fibrosis, were also target of this study. Our results show the presence of A2B receptor (A2BR) in GRX, and the regulation of extracellular hidrolysis of adenosine by TNF-α, through ecto-ADA activity improvement. Moreover, we demonstrate that extracellular adenosine does not modify the lipid synthesis in the GRX cells. The data still indicate that adenosine, at presence or absence of TNF-α, does not imply apoptosis, and that this cytokine does not induce the apoptotic bodies formation. The study shows that the production of NO was increased with TNF-α, with potentiation of this effect at the presence of adenosine, probably due to to A2BR, and not mediated by inosine, the product of adenosine hydrolysis. About the gelatinases, the treatment with adenosine decreases the MMP-9 activity in the absence, as well as in the presence of TNF-α, reversing the action of cytokine, at control group levels, with involvement of A2BR. However, the expression of MMP-9 was not affected by the treatment with adenosine, but the nucleoside reduces the effects of TNF-α on the expression of this gelatinase. About MMP-2, both treatment with adenosine and TNF-α, as well as the treatment with adenosine, in the presence of the cytokine, diminish the activity of gelatinase, without additive effects. The expression of MMP-2 mRNA increased in reponse to the treatments with adenosine and TNF-α, alone or in association, suggesting post-transcriptional mechanisms for the regulation of this enzyme. Our results clearly indicate that extracellular adenosine and TNF-α are involved in the regulation of NO production and actions of gelatinases. Moreover, this study demonstrated that adenosine do not act in phenotype conversion, through lipid synthesis, as well as do not stimulate apoptosis, toghether with TNF-α. This results and the existence of regulation of extracellular of adenosine in response to TNF-α further support that adenosine modulate some important aspects of HSC phisiology, maybe through A2B pathway.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A doenÃa de Parkinson (DP) à uma desordem neurodegenerativa, caracterizada pela destruiÃÃo dos neurÃnios nigroestriatais dopaminÃrgicos. O tratamento atual para esta doenÃa està restrito ao alÃvio sintomÃtico, porque atà o presente momento nÃo existem agentes capazes de inibir a degeneraÃÃo neuronal. Existem evidÃncias experimentais de que antagonistas de receptores A2A da adenosina poderiam ser Ãteis no tratamento de DP. Com a finalidade de investigar essa possibilidade, o presente trabalho demonstrou os efeitos da cafeÃna e do CSC (8-(3-chlorostyryl caffeine) no comportamento rotacional e nas alteraÃÃes neuroquÃmicas em ratos lesionados com 6-OHDA, como modelo da doenÃa de Parkinson. Os animais (ratos Wistar machos, 250-280g) foram tratados com cafeÃna (10 e 20 mg/kg, i.p.) diariamente durante 14 dias, iniciando 1h apÃs a lesÃo ou 7 dias, iniciando seis dias apÃs a lesÃo com 6-OHDA ou com CSC (1 e 5 mg/kg, i.p.) diariamente durante 7 dias, iniciando 6 dias apÃs a lesÃo com 6-OHDA, sozinho ou associado com L-DOPA (CSC 1 mg/kg, i.p. + L-DOPA 50mg/kg + Benzerazida 12,5 mg/kg, i.p.). Os resultados mostraram que houve um aumento significativo do nÃmero de rotaÃÃes induzidas por apomorfina nos animais lesionados com 6-OHDA (50 vezes) quando comparados aos animais falso operados. O tratamento com cafeÃna, principalmente durante 14 dias e o tratamento com CSC produziram uma recuperaÃÃo motora parcial com reduÃÃo do nÃmero de rotaÃÃes. A 6-OHDA provocou morte neuronal evidenciada pela reduÃÃo dos nÃveis de monoaminas (75-85%) quando comparadas ao lado contralateral. Nos grupos tratados com cafeÃna ou CSC sozinho ou associado com L-DOPA a reduÃÃo dos nÃveis de DA, 5HT e seus metabÃlitos foi menor. As concentraÃÃes dos aminoÃcidos glutamato e GABA foram significativamente aumentadas (3,8 e 3 vezes, respectivamente) no estriado de ratos lesionados. O CSC reverteu essas alteraÃÃes significativamente e foi observada uma potencializaÃÃo desses efeitos na associaÃÃo com L-DOPA. Os experimentos in vitro demonstraram que a cafeÃna e o CSC apresentaram um forte efeito neuroprotetor nas cÃlulas mesencefÃlicas de rato expostas a 6-OHDA. O tratamento com CSC ou cafeÃna aumentou significativamente o nÃmero de cÃlulas viÃveis apÃs a exposiÃÃo das cÃlulas a 6-OHDA, como foi demonstrado pelo teste do MTT. A exposiÃÃo das cÃlulas mesencefÃlicas a 6-OHDA aumentou os conteÃdos de nitrito e a peroxidaÃÃo lipÃdica, que retornaram a concentraÃÃes normais apÃs tratamento com CSC ou cafeÃna. AlÃm disso, a 6-OHDA reduziu o nÃmero de cÃlulas normais e aumentou o nÃmero de cÃlulas apoptÃticas e o tratamento com CSC ou cafeÃna reverteu esses efeitos da 6-OHDA, promovendo aumento do nÃmero de cÃlulas viÃveis e reduÃÃo do nÃmero de cÃlulas apoptÃticas. Houve uma reduÃÃo do nÃmero de microglias ativadas apÃs a exposiÃÃo das cÃlulas a cafeÃna e a 6-OHDA, o mesmo nÃo ocorreu apÃs a exposiÃÃo das cÃlulas ao CSC e a 6-OHDA. O tratamento com cafeÃna reduziu o aumento do nÃmero de astrÃcitos reativos induzidos pela 6-OHDA, enquanto o CSC nÃo apresentou esse efeito. Esses resultados mostraram que ambos, a cafeÃna e o CSC apresentaram aÃÃes neuroprotetoras em cÃlulas mesencefÃlicas de rato expostas a 6-OHDA. O presente trabalho mostrou que a cafeÃna e o CSC reverteram Ãs alteraÃÃes comportamentais e neuroquÃmicas da 6-OHDA, apresentando efeitos possivelmente benÃficos no tratamento da DP.
Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. Antagonists of the A2A subtype of adenosine receptor have emerged as a target for nondopaminergic antiparkinsonian agents. The present work showed the effects of caffeine and 8-(-3-chlorostyryl)-caffeine (CSC), A2A receptors antagonists, on behavior and biochemical alterations in 6-OHDA-lesioned rats, as a model of PD. Animals (male Wistar rats, 260-280 g) were injected daily with caffeine (10 and 20 mg/kg,i.p., 1h after 6-OHDA lesion for 14 days or six days after 6-OHDA lesion for 7 days), or CSC (1 and 5 mg/kg, i.p., 1h after 6-OHDA lesion for 7 days) alone or associated with L-DOPA (CSC 1 mg/kg, i.p. + L-DOPA 50mg/kg + Benzerazida 12,5 mg/kg, i.p., six days after 6-OHDA lesion for 7 days). Fourteen days after 6-OHDA, the animalsâ behavior was assessed by monitoring body rotations induced by apomorphine (3 mg/kg, i.p.). The results showed that the drastic increase in body rotation, induced by the 6-OHDA lesion, after the apomorphine challenge, was significantly (50 times) and dose-dependently reversed by CSC or caffeine. The decreased striatal levels of DA and metabolites, in the 6-OHDA-lesioned rats (75-85%), were blocked after caffeine or CSC alone or in association with L-DOPA treatment as well as the concentrations of NE, 5-HT and 5-HIAA. These effects were potentiated in 6-OHDA-lesioned animals treated with the association of CSC and L-DOPA. Concentrations of the amino acids glutamate and GABA were significantly increased (3.8 and 3 times, respectively) in the 6-OHDA-lesioned rat striatum. Similarly, CSC also reversed these alterations significantly. We also demonstrated protective effects against 6-OHDA-induced cytotoxicity in rat mesencephalic cells. Caffeine or CSC significantly increased the number of viable cells after their exposure to 6-OHDA, as measured by the MTT assay. While nitrite levels and lipid peroxidation in the cells were drastically increased by 6-OHDA, its concentration was brought toward normality after caffeine or CSC. 6-OHDA decreased the number of normal cells while increasing the number of apoptotic cells. Caffeine or CSC, significantly recovered the number of viable cells, and decreased the number of apoptotic cells, as compared to the group treated with 6-OHDA alone. Interestingly, while a significant lower number of activated microglia was seen after cells exposure to caffeine plus 6-OHDA, this was not the case after cells exposure to CSC plus 6-OHDA. While caffeine lowered the percentage of reactive astrocytes increased by 6-OHDA, CSC showed not effect. These results showed a strong neuroptrotection afforded by caffeine or CSC on rat mesencephalic cells exposed to 6-OHDA. In conclusion, we showed that CSC or caffeine reversed behavior and biochemical alterations, observed in the 6-OHDA-lesioned rats, pointing out to the potential benefit of A2A receptors antagonists as non-dopaminergic therapeutic targets for the treatment of PD.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
C. difficile toxin A (TxA) plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors (A2A ARs) attenuates inflammation and damage in many tissues. This study evaluated the effect of a new selective A2A AR agonist (4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester; ATL313) on TxA-induced enteritis in murine ileal loops. ATL313 (0.05-5 nM) and/or the A2A AR antagonist (ZM241385; 5 nM) or PBS were injected inside ileal loops immediately prior to challenge with TxA (1-10 mg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissue samples were collected for measurement of myeloperoxidase (MPO) content, evaluation of ADA activity, for histopathology and apoptotic immunohistochemistry (ApopTagÃ) and for assessment of TNF-α levels by ELISA. TxA (1-10 Âg/loop) significantly (p<0.05) increased volume/length and weight/length, reaching maximum values at 5Âg/loop dosage. ATL313 (5 nM) treatment significantly (p<0.05) reduced TxA-induced volume/length and weight/length, as well as prevented mucosal disruption and TxA-induced apoptosis. These protective effects were reversed by ZM241385 (5 nM), the A2A AR antagonist. ATL313 (5 nM) also reduced neutrophil infiltration, as measured by MPO content; reduced the toxin A-induced increase in ADA activity. Prior to the challenge with TxA, a systemic injection of fucoidin, but not PBS, also reduced tissue destruction and toxin A-induced increase in ADA activity. In conclusion, the A2A AR agonist ATL313 has a great antiinflammatory effect in TxA-induced mice enteritis, significantly reducing tissue destruction and ADA activity. In addition, our data suggested that TxA-induced increase in ADA activity and tissue damage in murine ileal loops are related to the neutrophil infiltration induced by this toxin.
A toxina A do Clostridium difficile (TxA) desempenha um importante papel na patogÃnese da diarrÃia induzida por antibiÃticos e na colite pseudomembranosa, uma condiÃÃo caracterizada por intensa secreÃÃo e inflamaÃÃo da mucosa. A estimulaÃÃo de receptores A2A da adenosina reduz a inflamaÃÃo e o dano tecidual. Neste estudo, avaliou-se o efeito de um novo agonista seletivo para receptores A2A da adenosina (metil Ãster do Ãcido 4-{3-[6-amino-9-(5-ciclopropilcarbamoil-3,4- dihidroxitetrahidrofuran-2-il)-9H-purin-2-il]prop-2-inil}piperidina-1-carboxÃlico; ATL313) na enterite induzida pela TxA em alÃas ileais de camundongos. O ATL313 (0,05-5 nM) e/ou o antagonista dos receptores A2A da adenosina (ZM241385; 5 nM) ou PBS foram injetados em alÃas ileais imediatamente antes da injeÃÃo de TxA (1-10 Âg/alÃa) ou PBS. As razÃes volume de secreÃÃo/comprimento da alÃa e peso/comprimento da alÃa foram calculadas 3h depois. Amostras de tecido foram coletadas para dosagem de atividade de mieloperoxidade (MPO), atividade de ADA, histopatologia, imunohistoquÃmica para apoptose (ApopTag_) e dosagem de TNF-a_ por ELISA. A injeÃÃo de TxA (1-10 Âg) nas alÃas ileais aumentou significativamente (p<0,05) as razÃes volume de secreÃÃo/comprimento da alÃa e peso/comprimento da alÃa com pico em 5Âg. O tratamento das alÃas com ATL313 (5 nM) reduziu significativamente (p<0,05) a secreÃÃo e o edema, preveniu a destruiÃÃo da mucosa e a apoptose induzidos por TxA. Tais efeitos protetores foram revertidos pelo antagonista dos receptores A2A de adenosina, o ZM241385 (5 nM). O tratamento com ATL313 (5 nM), reduziu ainda a infiltraÃÃo neutrofÃlica, avaliada pela dosagem de MPO, e reduziu o aumento da atividade de ADA induzidos pela TxA, bem como a dosagem de TNF-a no tecido das alÃas ileais. O prÃ-tratamento sistÃmico com fucoidina, mas nÃo com PBS, tambÃm reduziu o dano na mucosa e atividade de ADA no tecido das alÃas ileais tratadas com TxA. Assim, conclui-se que na enterite induzida pela TxA em camundongos, o agonista dos receptores A2A da adenosina (ATL313) possui um potente efeito antiinflamatÃrio, reduzindo consideravelmente a lesÃo tecidual e a atividade de ADA. Nossos resultados tambÃm indicam que o aumento da atividade de ADA e o dano tecidual induzido pela TxA em alÃa ileal de camundongos està relacionado com a infiltraÃÃo neutrofÃlica induzida por esta toxina.

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O Clostridium difficile à reconhecido por ser uma bactÃria nosocomial, levando a intensa inflamaÃÃo intestinal, quebra da barreira epitelial e diarreia. A produÃÃo de adenosina està aumenta durante eventos inflamatÃrios. O receptor de adenosina A2B (A2BR) à o mais expresso no intestino de humanos e camundongos. NÃs investigamos o efeito de exposiÃÃo Ãs TcdA e TcdB, a curto e longo prazo, em cÃlulas HCT-8 e cÃlulas epiteliais intestinais isoladas do ceco. CÃlulas HCT-8 foram expostas a TcdA ou TcdB (10 ng/ml) por 2, 6 e 24h. Foi usado o modelo de alÃa cecal e de infecÃÃo pelo bacilo em murinos para avaliar os efeitos da TcdA e da infecÃÃo pelo C. difficile, respectivamente. Foi demonstrado que HCT-8 e cÃlulas epiteliais intestinais isoladas do ceco naturalmente expressam altos nÃveis do receptor A2BR. TcdA e TcdB alteraram a morfologia celular, viabilidade e proliferaÃÃo e causaram aumento da expressÃo gÃnica de todos os subtipos de receptores de adenosina e das citocinas IL-6 e IL-8 em HCT-8. Em cÃlulas epiteliais intestinais isoladas do ceco, a TcdA significativamente causou um aumento do peso e volume/comprimento da alÃa cecal, escores histolÃgicos e infiltrado neutrofÃlico, medido por MPO, e tambÃm causou alteraÃÃes da expressÃo gÃnicas dos receptores de adenosina, tanto no modelo de alÃa cecal quanto na infecÃÃo pelo bacilo. O tratamento com PSB603, um antagonista do receptor A2BR, foi capaz de reverter os efeitos causados pelas toxinas do C. difficile. Nossos dados suportam a hipÃtese que as toxinas do C. difficile alteram a expressÃo dos receptores de adenosina e isso pode estar relacionado com os severos efeitos inflamatÃrios. NÃs concluimos que os receptores de adenosina tenham um papel importante na regulaÃÃo da inflamaÃÃo em cÃlulas epiteliais intestinais na infecÃÃo pelo C. difficile.
Clostridium difficile is recognized to be a nosocomial pathogen that causes intense intestinal inflammation, epithelial barrier disruption and diarrhea. Adenosine production is increased under inflammatory situations. The adenosine receptor A2B is the most expressed receptor in the human and mice intestine. We investigated the effect of short- and long-term exposure to TcdA and TcdB in HCT-8 cells and isolated cecum epithelial cells. HCT-8 cells were exposed to TcdA or TcdB (10 ng/ml) for 2, 6 and 24h. We used a murine cecal loop model and murine infection model to evaluate the effects of TcdA and C. difficile infection, respectively. We demonstrated that HCT-8 and isolated intestinal cecum epithelial cells naturally express high levels of A2BR receptors. TcdA or TcdB alters the cell morphology, viability and proliferation pattern and caused gene expression increase of all AR subtypes in HCT-8. In isolated cecum epithelial cells, TcdA significantly (p<0.05) increased volume/length, weight/length, histopathology scores, neutrophil infiltration, as measured by MPO content, and induced an altered gene expression increase of all AR subtypes. PSB603 (10 nM) treatment significantly (p<0.05) reduced TcdA-induced tissue damage. Our findings support the hypothesis that Clostridium difficile toxins affect adenosine receptor expression and this action may be related to their severe inflammatory effect. We concluded that adenosine receptors may play a crucial role in regulating the inflammatory system in intestinal epithelium during C. difficile infection.

Endothelial inflammation leading to vascular dysfunction is a major contributor to the development of atherosclerosis. The release of adenosine at sites of inflammation represents an endogenous mechanism for limiting excessive inflammation and tissue damage. The majority of the anti-inflammatory effects of adenosine are mediated by signalling through the A2AAR and activation of the A2AAR has been shown to be protective in numerous models of inflammatory disease. Little is known about the molecular mechanisms behind these effects. However, in vitro studies using cultured endothelial cells indicate that signalling through the A2AAR can suppress activation of the NF kappa B and JAK/STAT proinflammatory signalling pathways. NF kappa B appears to be primed for activation in atherosclerosis-prone regions of the aorta indicating that suppression of NF kappa B signalling may protect against the development of atherosclerosis. In this study, the role of the A2AAR in regulating NF kappa B and JAK/STAT signalling pathway activation in the aorta was studied using A2AAR-deficient mice subjected to an LPS-induced model of septic shock. In response to LPS treatment, these mice displayed markedly elevated plasma levels of the pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and GMCSF compared to wild-type mice. Consistent with this finding, heightened activation of the NF kappa B and JAK/STAT pathways was detected in aortic protein samples from A2AAR-deficient mice as demonstrated by increased levels of the phosphorylated forms of I kappa B alpha and STAT1. However, expression of the NF kappa B and STAT1-regulated genes ICAM-1, VCAM-1 and TAP-1 was unaffected indicating the involvement of compensatory negative feedback mechanisms. These findings confirm a role for the A2AAR in regulation of pro-inflammatory signalling in the aorta. Further analysis of mechanisms which mediate this response may reveal new targets for therapeutic intervention to suppress inflammation in inflammatory disorders such as atherosclerosis. While the wide range of anti-inflammatory and tissue-protective responses elicited by the A2AAR have been well documented, the molecular regulation of the A2AAR has been less well studied. The presence of several serine and threonine residues in the extended C-terminal tail of the A2AAR suggests that it may be regulated by phosphorylation events occurring in this region. Indeed, the canine A2AAR is phosphorylated in response to PKC activation. Interestingly, several proteins have recently been identified as being able to interact with the C-terminal tail of the A2AAR. However, how these interactions are regulated is not known. One of the aims of this study was to characterise phosphorylation of the human A2AAR and to determine whether this could provide a means for regulating the binding of C-terminal interacting proteins. This was examined using human umbilical vein endothelial cells infected with recombinant adenovirus encoding the human A2AAR. It was found that phosphorylation of the human A2AAR could be induced in HUVECs by treatment with PMA or by stimulation of endogenous histamine H1 receptors. This was due to activation of PKC, as phosphorylation was inhibited by the PKC inhibitor GF109203X and by depletion of PKC following chronic treatment with PMA. Treatment of cells with the calcium-chelating agent BAPTA/AM did not inhibit PMA-induced phosphorylation indicating that a calcium-insensitive isoform of PKC was responsible. Meanwhile an siRNA-mediated gene silencing approach confirmed that PKC epsilon was not responsible indicating the involvement of either PKC delta or PKC theta. Previously reported interactions between the A2AAR and TRAX and 14-3-3 tau were confirmed in vitro by GST pull-down assay. Binding of 14-3-3 tau to the A2AAR appeared to be unaffected by treatment of HUVECs with PMA. However, A2AAR complex formation with TRAX was significantly reduced in samples from PMA-stimulated cells. These findings indicate that PKC-mediated phosphorylation may represent a means of regulating which proteins can interact with the C-terminal tail of the A2AAR. This may allow the A2AAR to initiate distinct signalling pathways depending on the cellular context in order to achieve the appropriate response.

The research reported in this Ph.D. thesis, though it has to be deepened, furthers our understanding about the ARs. New AR ligands were developed preferentially belonging to the aminopyridine-3,5-dicarbonitrile series whose structure-activity relationships have been till now poorly investigated. In fact, most of the compounds reported in the literature are included in patent documents. In this work, many derivatives were synthesized and biologically evaluated in binding and functional assays in order to assess affinity and selectivity towards a specific AR subtype, but also to evaluate their pharmacological profile. The data obtained confirmed the peculiarity of this series whose derivatives are not only characterized by a wide range of affinities but interestingly endowed with different degree of efficacies at the different ARs. Based also on molecular modelling studies two set of compounds, named DCP1 and DCP2B were designed preferentially targeting the A1 and A2B AR, respectively. Most of the compounds belonging to the DCP1 set are endowed with high affinity and good selectivity at the hA1 AR. Some selected derivatives showed also an inverse agonist profile with the only exception of compound 15 which emerged as partial agonist. Thus, further modification at the level of the thiazole moiety will be planned in order to evaluate the importance of this substituent for obtaining hA1 AR agonists. Moreover, when evaluated in an oxaliplatin-induced neuropathic pain model in mice, compound 1 (dual A1 inverse agonist/A2A antagonist) resulted to revert allodynia at very low doses after oral administration. This effect is similar to that exerted by both caffeine and other A1/A2A antagonists as well as A1 AR agonists in reverting hyperalgesia and in antinociception. Stability studies in mouse and human plasma on 1 confirmed no significant chemical modification of the compound for the whole duration of the assay. Thus, to clarify this paradoxical data, other molecules belonging to this set, endowed with similar degree of affinities and efficacies with respect to 1, will be tested in this in vivo assay, also to evaluate their potentiality for neuropathic pain treatment. The results obtained on the DCP1 set, especially in the in vivo assay, suggested that the amino-3,5-dicyanopyridine scaffold could be of interest for obtaining non-nucleoside AR agonists as to prevent two serious problems hampering the development of classical adenosine-like agonists: the low oral bioavailability and the short half-life due to the presence of the typical ribose (or ribose-mimicking) moiety. DCP2B set is composed of compounds preferentially targeting the hA2B receptor. Most of them showed to behave as partial agonist, with the best results concerning activity and selectivity of compound 37, which is three fold more potent than the reference agonist BAY60-6583. This result can be considered a real breakthrough since BAY60-6583 is absolutely the sole potent and selective hA2B AR agonist reported in literature. Moreover, it is noteworthy that derivatives 66 and 67, showing high activity at the hA2BAR but scarce selectivity, emerged as the only two amino-3,5-dicyanopyridines reported till now which are endowed with a full agonist profile. With this in mind, a study to clarify the structural requirements which are important in determining the A2B pharmacological profile of these compounds is currently on going. Thus, identification of new selective hA2B agonists could be of help in deepening the medicinal chemistry and the pharmacology related to this still unknown AR subtype. In fact, compound 37 was used as pharmacological tool in an oligodendrocyte precursor cell (OPC) differentiation assay in order to clarify whether the A2B receptor represents a promising target to modulate endogenous remyelinization process in multiple sclerosis patients. Compound 37 inhibits OPC differentiation into oligodendrocytes at lower concentration than those used for BAY60-6583 demonstrating the role of A2B AR in this important physiological process. Moreover, docking studies were performed on selected DCP1 and DCP2B derivatives at A1 and A2B homology receptor models, respectively, showing the best binding poses for compounds 15 and 37 and the most important residues involved in the binding at these two AR subtypes. It seems to clarify the role of R4 and R6 substituents, containing atoms or groups able to engage hydrogen-bonding interactions with the receptors, in modulating affinity and efficacy of the amino-3,5-diyanopyridine derivatives. Moreover, since some affinity was also achieved at the hA2A and hA3 AR with some of the compounds detailed in this thesis, it may be possible that this heterocycle core could be also manipulated to enhance affinity and selectivity for these subtypes. In fact, neither A2A nor A3 agonists belonging to the amino-3,5-dicyanopyridine series has been ever designed. Preliminary studies on the newly synthesized 7-amino-2-phenylpyrazolo[1,5-c]pyrimidine-4-carbonitrile system, derived from molecular complications of the amino-3,5-dicyanopyridine core, were reported. Biological results on a small set of compounds, that bind with good affinity the hA1 AR, indicated that this scaffold suitably decorated could generate new AR ligands.

Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. The acute injury, characterized by a massive increase of extracellular glutamate levels, is followed by activation of resident immune cells and production or activation of inflammation mediators. Although after ischemia precocious activation of immune cells may be neuroprotective and supportive for regeneration, protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. In this thesis I investigated on the putative protective effects of ligands at histamine and adenosine receptors. Hstamine is recognized as an important neurotransmitter or neuromodulator in the Central Nervous System (CNS). Numerous studies have indeed suggested that histamine acting on the H1 and H2 receptors may play a protective effect during ischemia while no informations there were on the role of the H4 receptors that is considered the main immune system histamine receptor with a pro-inflammatory role in the periphery while its role in brain immune responses was scarcely studied. In the experimental model of focal cerebral ischemia induced by occlusion of the middle cerebral artery (MCAo) in rats, the levels of histamine evaluated by microdialysis increase in the ischemic areas. The histamine H4 receptor is predominantly expressed in cell types of immune system where it is involved in the regulation of immunological and inflammatory responses, and in numerous areas of the CNS including cortex and striatum. Therefore a first aim of my thesis was to investigate on the putative neuroprotective effects of the selective H4 receptor antagonist, JNJ7777120. Chronic treatment with JNJ7777120, significantly protects from the neurological deficit 1, 5 and 7 days after tMCAo (p<0.001 at each time point) and significantly reduced the body weight loss at 5 and 7 days after tMCAo with respect to vehicle-treated rats (respectively p<0.05; p<0.01). Seven days after the ischemic insult, JNJ7777120 reduces the volume of the ischemic cortical damage (p<0.0005) and the volume of the ischemic striatal damage (p<0.0001). Moreover, H4 antagonist reconstitutes the cytoarchitecture of ischemic cortex and striatum and decreases the number of heterochromatic small nuclei 7 days after transient ischemia. JNJ7777120, significantly reduces the number of IBA1+ (specific for microglia) and GFAP+ (specific for astrocytes) cells in the ischemic core and boundary zones of striatum and cortex with respect to vehicle-treated rats, and also decreases the plasma levels of the proinflammatory cytokines, IL-1β and TNF-α, while increases the levels of IL-10 regulatory cytokine with anti-inflammatory action , 7 days after tMCAo. Two days after ischemia, JNJ7777120 reduces granulocytes (evaluated as HIS-48+ cells) infiltration . Our results have demonstrated that the selective antagonist of histamine H4 receptor, JNJ7777120, systemically and chronically administered after ischemia, reduces the ischemic brain damage and improves the neurological deficit, by controlling a secondary inflammatory damage after brain ischemia Results demonstrate that the histamine H4 receptor represents a new interesting target after brain ischemia. Extracellular adenosine concentration dramatically increases during cerebral ischemia and a protective role is recognized to adenosine by acting on A1 receptors. However the use of adenosine A1 agonists is hampered by peripheral and central side effects. Most recent evidence indicated that the adenosine A2A receptor subtype is of critical importance in stroke. An overexpression of A2A receptors was found in neurons and microglia of the striatum and cortex after focal ischemia induced by permanent MCAo. Previous results from the laboratory where I carried out my thesis work have demonstrated that adenosine A2A receptor antagonists repeatedly administered after brain ischemia have protected from the ischemic damage evaluated 24 hours thereafter likely reducing early acute excitotoxic phenomena. Most recently, the question has been raised if A2A receptor continuous blockade maintain protection at a more distant time after ischemia. A second aim of my thesis was therefore to investigate if the selective A2A receptor antagonist, SCH58261 maintains the protective effects 7 days after ischemia in a model of transient cerebral ischemia in the rat. The adenosine A2A receptor antagonist, SCH58261, administered twice/day for 7 days protects from neurological deficit 1 day after tMCAo (p<0.01), but no more after 5 and 7 days. Moreover, seven days after tMCAo, SCH58261 does not protect ischemic areas from damage and does not ameliorate myelin organization into the ischemic striatum. Two days after tMCAo, SCH58261 does not reduce blood cell infiltration into ischemic striatal and cortical tissue. The lack of protection by the A2A receptor antagonist 7 days after ischemia could be explained by with the fact that the early protective effect from acute excitotoxicity is overwhelmed by the subsequent damage brought about by massive cell infiltration and neuroinflammation. Indeed neuroinflammation is now recognized as a predominant mechanism of secondary progression of brain injury after ischemia. Clarification of the time-limit in which A2A receptor antagonism is protective after brain ischemia might let to devise a correct therapeutic strategy with A2A receptor antagonists. Few studies are present in literature on the role of A2B receptors in brain ischemia. An overexpression of A2B receptors occurs on endothelial cells, neurons and astrocytes 24 hours after tMCAo in the rat. To date there are no evidences in literature on the protective effects of A2B receptor agonists at more distant times from ischemia when a defined neuroinflammation develops. A further aim of my thesis was to investigate, in the model of transient cerebral ischemia in the rat, the putative protective effects of the A2B receptor agonist, BAY 60-6583 7 days after ischemia, when a clear inflammatory response has developed. Treatment with adenosine A2B receptor, BAY 60-6583, chronically administered, improves the neurological deficit evaluated 1 and 5 and up to 7 days after tMCAo (p<0.001-0.02), but does not protect from the body weight loss. Seven days after ischemia, BAY 60-6583 significantly reduces the infarct area within the cortex (p<0.04) and causes a trend towards a reduction of damage also in striatum. Seven days after transient ischemia, BAY 60-6583 has reconstituted the cortical and striatal cytoarchitecture. Both peripheral antiadhesion and central antinflammatory effects could account for protective effect of A2B agonists but further experiments will investigate on the intimate brain cell transduction mechanisms involved in the A2B protective effects after transient ischemia.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that leads to destruction of articular cartilage and subchondral bone at the synovial joints. Clinically, RA is characterized by swelling, tenderness and destruction of synovial joints, which results in severe disability and premature mortality. In the RA disease state, inflammation in the synovial compartment is regulated by a complex cytokine and chemokine network, including tumor necrosis factor α (TNFα), which has been clinically demonstrated a key mediator of RA pathogenesis. TNFα can be found in elevated levels in the synovial fluid and serum of RA patients and the role of the cytokine in both the inflammation and bone destruction of RA suggests it is important in the understanding of disease progression as well as the development of therapeutic targets. Many of the biological processes that mediate RA, including bone turnover and cartilage resorption, involve signaling pathways that are mediated by adenosine and its receptors. The A2B adenosine receptor (A2BAR) is highly expressed in the synoviocytes of RA patients and the receptor has a similar expression profile in humans and mice. The goal of this thesis was to use a mouse model of RA to understand how the A2B adenosine receptor modulates TNFα and other destructive enzymes that contribute to the progression of the disease. A collagen antibody-induced arthritis (CAIA) mouse model was used to determine the effect of A2BAR ablation on systemic and joint-specific TNFα expression. Comparable arthritic conditions were observed in CAIA mice of both A2BAR knockout (KO) and wild-type (WT) genotypes and the absence of the A2BAR gene did not result in any observable differences in the gross arthritic state created in each genotype. Immunohistochemistry analysis of TNFα expression in mouse paws revealed that paw joints from CAIA A2BAR KO mice exhibited more robust TNFα staining compared to CAIA WT specimens of the same treatment duration. ELISA analysis of the serum showed that only CAIA A2BAR KO mice had greater serum production of TNFα at day 10 after induction of arthritis. TNFα and matrix metalloproteinase-9 mRNA expression were also elevated in KO CAIA knee joints in comparison to WT CAIA knee joints; however, WT CAIA mice were found to have higher levels of aggrecanase mRNA compared to KO mice. These results suggest that while the loss of A2BAR activity leads to a hyper-inflammatory state, the A2B adenosine receptor alone is not responsible for the progressive inflammation of the synovial joints associated with rheumatoid arthritis.

Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
A Hipertensão Arterial Pulmonar (HAP) é uma doença complexa e multifactorial que induz uma sobrecarga no coração, devido ao aumento da resistência da circulação pulmonar. Neste cenário de aumento de resistência da circulação pulmonar, o lado direito do coração fica sobrecarregado, conduzindo à remodelação tissular do miocárdio e, eventualmente, falência do ventrículo direito (VD). A adenosina (ADO) é uma molécula de sinalização que é ubíqua e capaz de modular a função cardíaca mediante a interação com vários subtipos de recetores (A1AR, A2AAR, A2BAR e A3AR). Tem sido descrito que o bloqueio do A2BAR protege o pulmão da inflamação e fibrose, em doenças pulmonares intersticiais, embora o papel exato deste recetor sobre a progressão da remodelação ventricular na HAP, permaneça indefinido e largamente inexplorado. Neste contexto, o presente estudo pretendeu investigar o papel do A2BAR na disfunção cardíaca secundária à HAP realizando estudos in vitro com aurículas isoladas, tiras de VD e fibroblastos cardíacos (FC) em cultura. A HAP foi induzida em ratazanas Wistar machos que receberam aleatoriamente uma única injeção subcutânea de monocrotalina (grupo MCT) ou veículo (grupo CTRL). Os estudos ecocardiográficos, hemodinâmicos, miográficos (aurículas espontaneamente contráteis e tiras de VD eletricamente estimuladas) e análises morfométricas e histológicas foram realizados 21 a 25 dias após a administração de monocrotalina ou solução salina. Foram também realizados estudos de proliferação e produção de colagénio tipo I em culturas primárias de FC isolados a partir de VD. Observou-se que o A2BAR não parece modular a frequência nem a capacidade contráctil das aurículas a contrair espontaneamente, em ambos os grupos experimentais. A ativação seletiva do A2BAR, em tiras de VD, com o BAY 60-6583 (0.01–10 μM) foi desprovida de efeito inotrópico tanto em ratazanas CTRL como MCT. Contudo, o prétratamento das tiras de VD com o antagonista A2BAR, PSB 603 (100 nM), converteu o efeito inotrópico negativo do agonista não seletivo da ADO, NECA (0.01–100 μM), num efeito inotrópico positivo no grupo MCT, ao contrário do que foi observado em ratazanas CTRL. O efeito do PSB 603 (100 nM) não foi mimetizado pela inibição de algumas vias de sinalização classicamente associadas à ativação A2BAR, como a ciclase do adenilato, a fosfolipase C e a proteína cinase C. Os estudos de imunofluorescência revelaram que os cardiomiócitos de VD de ratazanas CTRL e MCT apresentavam pequenas quantidades de A2BAR. Além disso, o miocárdio ventricular direito de animais com HAP apresentava infiltrados celulares imunoreativos para A2BAR nos espaços intersticiais. Estes infiltrados celulares exibiram também imunoreatividade positiva para marcadores celulares como a vimentina (fibroblastos) e CD11b (macrófagos/monócitos). No que concerne aos FC isolados de ratazanas adultas dos grupos CTRL e MCT, estes apresentaram características de miofibroblastos já que possuiam imunorreatividade contra o domínio do recetor discoide 2 (DDR2), vimentina e actina muscular lisa (α-SMA). A incubação das culturas de FC com NECA (10 μM) aumentou a sua proliferação/viabilidade bem com a produção de colagénio do tipo I. O efeito promotor da proliferação observado com a NECA foi significativamente atenuado na presença do antagonista A2BAR, PSB 603 (100 nM), em animais MCT. O mesmo não foi observado nos animais CTRL, sugerindo que a ativação A2BAR favorece a proliferação dos FC induzida pela NECA nos animais com HAP. Resumindo, os dados mostraram que a ativação do A2BAR contribui para a diminuição da contratilidade do VD nos animais com HAP. Este efeito poderá estar relacionado com a infiltração dos espaços intersticiais do miocárdio do VD dos animais com HAP por fibroblastos e células inflamatórias que exibem imunoreactividade contra o A2BAR. A ativação destes receptores potencia a proliferação dos FC e a fibrose miocárdica, podendo ainda os infiltrados celulares detectados serem responsáveis pela libertação de mediadores químicos com características inibidoras da performance contrátil dos cardiomiócitos em animais com HAP. O impacto clínico da manipulação terapêutica dos receptores A2BAR localizados nos fibroblastos e células inflamatórias dos insterstícios miocárdicos na progressão da insuficiência cardíaca direita em indivíduos com HAP carece de estudos complementares de longa duração in vivo, incluindo ensaios hemodinâmicos, ecocardiográficos, morfométricos e histológicos.
Pulmonary arterial hypertension (PAH) is a complex and multifactorial disease that overloads the heart by increasing the resistance of pulmonary circulation. In this scenario of higher pulmonary circulatory resistance, the right side of the heart becomes overloaded, leading to myocardial tissue remodelling and eventually right ventricle (RV) failure. Adenosine (ADO) is a ubiquitous signalling molecule that is capable of modulating cardiac function through the interaction of receptor subtypes (A1AR, A2AAR, A2BAR and A3AR). It has been described that blockade of A2BAR protects lung from inflammation and fibrosis in interstitial pulmonary diseases, although the precise role of this receptor on progression of ventricular remodeling to heart failure in PAH remains inconclusive and largely unexplored. In this context, this study was designed to investigate the role of A2BAR in cardiac dysfunction secondary to PAH, by performing in vitro studies with isolated atria, RV strips and cardiac fibroblasts (CF) in culture. PAH was induced in male Wistar rats that were randomized to receive a single subcutaneous injection of MCT (MCT group) or vehicle (CTRL group). Echocardiographic, hemodynamic, myographic studies (spontaneously beating atria, RV strips electrically paced at 2 Hz-frequency), and morphometric and histological analysis were performed 21-25 days after MCT or saline administration. Proliferation and type I collagen studies of primary cultures of CF isolated from RV were also carried out. We observed that A2BAR did not seem to modulate the rate or tension of spontaneously beating atria in both experimental groups. In RV strips, the selective activation of the A2BAR with BAY 60-6583 (0.01–10 μM) was devoid of effect in inotropy in both CTRL and MCT rats. However, pre-treatment of RV strips with the A2BAR antagonist, PSB 603 (100 nM), converted the negative inotropic effect of the non-selective ADO receptor agonist, NECA (0.01–100 μM), into a mild positive inotropic action in the MCT group, contrary to what was observed in CTRL rats. The effect of PSB 603 (100 nM) was not mimicked by inhibiting some of the signalling pathways classicaly associated to A2BAR activation, such as adenylate cyclase, phospholipase C and protein kinase C. Immunofluorescence studies revealed that RV cardiomyocytes from CTRL and MCT rats expressed small amounts of A2BAR. Furthermore, RV myocardium of PAH animals exhibited A2BAR immunoreactive cell infiltrates at interstitial spaces. These cell infiltrates also exhibited positive immunoreactivity against vimentin (fibroblasts) and CD11b (macrophages/monocytes) cell markers. With respect to CF isolated from adult rats of CTRL and MCT groups, these had features of myofibroblasts since they possessed immunoreactivity against discoidin domain receptor 2 (DDR2), vimentin and α-smooth muscle actin (α–SMA). Incubation of CF cultures with NECA (10 μM) increased the viability/proliferation as well as type I collagen production. The proliferation promoter effect observed with NECA was significantly attenuated in the presence of A2BAR antagonist, PSB 603 (100 nM), in MCT animals. The same was not observed in CTRL animals, suggesting that A2BAR activation favours CF proliferation induced by NECA in PAH rats. In summary, data showed that A2BAR activation contributes to the reduction of RV contractility in PAH animals. This effect may be related to infiltration of RV myocardial interstitial spaces of PAH animals with fibroblasts and inflammatory cells that exhibited immunoreactivity against A2BAR. Activation of these receptors enhances CF proliferation and myocardial fibrosis. Detected cell infiltrates may be responsible for the release of chemical mediators with inhibitory characteristic of contractile performance of cardiomyocytes in PAH animals. The clinical impact of therapeutic manipulation of A2BAR located on fibroblasts and inflammatory cells, in the myocardial interstices, in progression of right heart failure in PAH patients, lacks further in vivo long-term studies, including hemodynamic, echocardiographic, morphometric and histological studies.
Work supported by FCT through projects FCOMP-01-0124-FEDER-028726 (FEDER, COMPETE, PTDC/DTP-FTO/0802/2012) and PEst-OE/SAU/UI0215/2014

Obesity is a significant health care problem, affecting more than one third of the United States population and is an important risk factor for Type 2 Diabetes Mellitus (T2D). Adipose tissue expansion results in the recruitment and accumulation of macrophages, which secrete proinflammatory cytokines that impair insulin signaling. Adenosine regulates inflammation by signaling through G-protein coupled receptors (GPCRs), such as the A2b adenosine receptor (A2bAR). Recently a role for adenosine receptors has been described in the differentiation of osteoblasts and adipocytes. This thesis tests the hypothesis that the A2bAR regulates adipose tissue dynamics at the level of preadipocyte differentiation and macrophage inflammation. This thesis showed that activation of the A2bAR inhibited preadipocyte differentiation. A2bAR-induced adipocyte inhibition was dependent on the expression of Krüppel-like factor 4 (KLF4), which is important for stem cell maintenance and renewal. A2bAR knockdown enhanced adipogenesis in vitro and A2bAR knockout (KO) mice had more adipocytes as compared to wild type (WT) mice, suggesting enhanced adipogenesis in the absence of the A2bAR. The translational potential of this work is strengthened by the previous finding of elevated A2bAR expression in adipose tissue of obese individuals as well as our new finding of a close correlation between the expression of A2bAR and KLF4 in adipose tissue of obese individuals. A2bAR KO mice have impaired insulin resistance, in part due to reduced levels of insulin receptor substrate-2 (IRS-2). Proinflammatory cytokines have been shown to reduce IRS-2 levels. Given the role of the A2bAR in regulating inflammation, the contribution of A2bAR signaling in macrophages to insulin resistance was elucidated. Transgenic mice that express A2bAR only in macrophages were generated. Intriguingly, restoration of A2bAR signaling in macrophages ameliorated insulin resistance, glucose tolerance, and fat and liver tissue insulin signaling. As expected, tissue and plasma proinflammatory cytokine levels were reduced to that of WT mice. This suggested that the protective effect of A2bAR signaling on insulin resistance was due in large part to A2bAR control of macrophage cytokine expression. This thesis highlights the importance of A2bAR signaling in adipogenesis and in regulating inflammation in the setting of obesity and T2D.

A hipertensão arterial pulmonar (HAP) é uma doença progressiva e potencialmente fatal que afeta a vasculatura pulmonar e, consequentemente, o coração. Esta doença cardiopulmonar é caracterizada por uma ampla variedade de fatores desencadeadores iniciais que promovem a remodelação da vasculatura pulmonar, aumentando a sua resistência ao fluxo sanguíneo. Este estado de elevada resistência circulatória pulmonar sobrecarrega o ventrículo direito (VD). A sobrecarga contínua exercida pelo incremento da pressão da artéria pulmonar é agravada pelo aumento da massa muscular (remodelação do VD) no sentido de aumentar a sua função contráctil para vencer a resistência. Frequentemente, o VD é incapaz de suportar a sobrecarga contínua a que é submetido e transita para um estado de insuficiência cardíaca, causando a morte do paciente. A adenosina (ADO) é um nucleósido purínico ubíquo capaz de modular a função cardíaca. A ADO atua como uma molécula sinalizadora através da ativação de quatro subtipos de recetores: A1AR, A2AAR, A2BAR e A3AR. Entre estes, os recetores A2AAR e A2BAR são os subtipos maioritariamente expressos em fibroblastos cardíacos (FCs), o tipo celular mais abundante no coração. Estas células apresentam um papel relevante na hipertrofia e fibrose do VD em pacientes com HAP, contribuindo para a disfunção e insuficiência deste órgão. Suspeita-se que a estimulação dos recetores A2AAR e A2BAR induzida pela ADO pode estar envolvida nas propriedades hiperproliferativas e profibróticas dos FCs do VD em doentes com HAP. Neste contexto, o presente estudo pretendeu investigar o papel destes dois recetores na proliferação e produção de colagénio do tipo I por FCs em cultura, isolados do VD de ratazanas adultas com HAP. A HAP foi induzida, aleatoriamente, em ratazanas Wistar machos através de uma injeção subcutânea única de monocrotalina (MCT; grupo MCT) ou veículo (NaCl 0.9%; grupo CTRL). As ratazanas foram eutanasiadas 21 a 25 dias após a administração de MCT ou solução salina. Os estudos de proliferação e produção de colagénio tipo I foram realizados em subculturas primárias de FCs do VD mantidos em cultura durante 28 dias. Amostras de tecido ventricular direito (VD) e esquerdo (VE) foram, ainda, colhidas para sequenciação do RNA, que por sua vez nos permitiu analisar a expressão génica diferencial entre ratazanas CTRL e MCT. Observou-se que o bloqueio do recetor A2BAR com PSB 603 (100 nM) reduziu a proliferação induzida pelo análogo estável da ADO, NECA (10 µM), nos FCs isolados de ratazanas MCT. A ativação e/ou o bloqueio do recetor A2AAR respetivamente com CGS 21680 (3 e 10 nM) e SCH 442416 (100 nM), não alterou a viabilidade/proliferação celular nem a produção de colagénio tipo I pelos FCs de ambos os grupos de animais. Relativamente à análise da expressão génica diferencial, os nossos dados mostraram que a remodelação, disfunção e insuficiência do VD em ratazanas MCT estão relacionadas com a sobreexpressão de vários genes codificantes de proteínas que estão envolvidas na progressão do ciclo celular, na replicação do DNA e nas interações matriz extracelular-recetores, mas também na via de sinalização PI3K-AKT. Além disso, embora o VE tenha sido considerado intrinsecamente normal no contexto desta doença, o estudo genético permitiu observar alterações significativas da expressão génica que poderão ser funcionalmente relevantes durante o desenvolvimento e progressão da HAP.
Pulmonary arterial hypertension (PAH) is a progressive life-threatening disorder that affects the pulmonary vasculature and consequently the heart. This cardiopulmonary disease is characterized by a wide variety of initial triggering insults which promote remodeling of the pulmonary vasculature, thereby increasing pulmonary vascular resistance. This state of higher pulmonary circulatory resistance overloads the right ventricle (RV) of the heart, which responds to the sustained pressure overload by accumulating muscle mass (RV remodeling) to enhance its contractile function. In many cases, the RV is unable to cope with the increase in load and heart failure develops, thus causing the patient’s death. Adenosine (ADO) is a ubiquitous purine nucleoside that modulates cardiac function. It acts as a signaling molecule through its interaction with four adenosine receptor (AR) subtypes: A1AR, A2AAR, A2BAR, and A3AR. Among these, A2AAR and A2BAR are highly expressed in cardiac fibroblasts (CFs), the most prevalent cell type in the heart. Since these cells provide a great contribution to the hypertrophic and fibrotic conditions of RV in PAH patients, ultimately leading to right heart dysfunction and failure, ADO-induced stimulation of A2AAR and A2BAR may mediate the hyperproliferative and profibrotic properties that CFs show in the RV of these patients. In this context, this study was designed to investigate the role of both ARs in the proliferation and type I collagen production by cultured CFs isolated from the RV of adult PAH rats. PAH was induced in male Wistar rats that were randomized to receive a single subcutaneous injection of MCT (MCT group) or vehicle (NaCl 0.9%; CTRL group). Rats were euthanized after 21-25 days since MCT or saline administration. Proliferative and type I collagen studies were performed on primary subcultures of RV CFs allowed to grow for 28 days. In addition, RV and left ventricle (LV) tissue samples were also collected to perform a RNA sequencing approach, which allowed us to achieve a differential gene expression analysis between CTRL and MCT rats. We observed that blockade of A2BAR with PSB 603 (100 nM) in cultured CFs from MCT rats decreased the proliferation of these cells induced by the non-selective AR agonist, NECA (10 µM). Furthermore, stimulation and blockade of A2AAR with CGS 21680 (3 and 10 nM) and SCH 442416 (100 nM), respectively, were devoid of effect in cell viability/proliferation and type I collagen production by cultured CFs from both animal groups. Regarding the differential gene expression analysis, our data showed that the RV remodeling, dysfunction and failure in MCT rats are related to the upregulation of several genes encoding proteins that are involved in the cell cycle progression, DNA replication, and extracellular matrix-receptor interactions, but also in the PI3K-AKT signaling pathway. Moreover, although the LV has been considered intrinsically normal in the context of PAH, we also demonstrated that it undergoes significant gene expression changes which may be functionally relevant during PAH development and progression.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Adenosine is a neuromodulator able to control the balance between neuronal excitation and inhibition, throughout the central nervous system (CNS). Adenosine acts onto a G protein–coupled receptors (GPCRs) subfamily called P1 adenosine receptors: A1R, A2AR, A2BR and A3R. Adenosine type 2A receptor (A2AR) is highly expressed in striatum, whereas in other brain regions A2AR is expressed at lower levels. One of these regions is the hippocampus, a central brain region enrolled in learning and memory processes as well as spatial recognition. Moreover, it was discovered that A2AR modulate reference memory. Additionally, A2AR can facilitate synaptic transmission since it can fine-tune other neuromodulatory systems, by controlling neurotransmitter release and modulating metabotropic or ionotropic receptors. It was also described that A2AR can interact with NMDA receptor (NMDAR), that is an ionotropic receptor. Therefore, given the particular importance of NMDAR in the hippocampal neurotransmission phenomena, in this work, we explore how A2AR can modulate NMDAR-dependent currents in hippocampal Schaffer collaterals - CA1 synapses. We showed that exogenous activation of A2AR, with its selective agonist CGS21680, decreases NMDAR-dependent evoked excitatory postsynaptic currents (eEPSCs) and that this effect is no longer observed in the presence of the selective A2AR antagonist, SCH58261. However, the superfusion of SCH58261 alone also decreases NMDAR-eEPSCs, suggesting that A2AR may exert a tonic control of these currents in CA1 pyramidal cells. Finally, this study revealed that our slice preparations contained high levels of endogenous adenosine which, once removed, does not prevent the A2AR antagonist-induced decrease in NMDAR-eEPCSs in SC-CA1 synapses. Our main hypothesis to explain these results is based in the possibility of existing two sub-populations of A2AR that may have antagonistic effects upon NMDA-dependent currents. Nevertheless, this hypothesis and mechanisms remain to be clarified, which can be the foundation for future work.
A adenosina é um neuromodulador capaz de controlar o balanço entre excitação e inibição ao longo de todo o sistema nervoso central. Este controlo é feito através da activação da subfamília de receptores acoplados a proteínas G (GPCRs), os receptores de adenosina P1: A1R, A2AR, A2BR and A3R. O receptor de adenosina do tipo 2A (A2AR) é abundantemente expresso no estriado e é menos expresso noutras regiões do cérebro. Uma destas regiões é o hippocampo, uma região cerebral central envolvida nos processos de aprendizagem, memória e reconhecimento espacial. Foi descoberto que os A2AR podem modular a memória de referência. Além disso, os A2AR estão maioritariamente descritos como facilitadores da transmissão sináptica, sendo que as suas principais funções são o refinamento de outros sistemas neuromodulatórios, o controlo da libertação de neurotransmissores e ainda a modulação da actividade de outros receptores, quer metabotrópicos quer ionotrópicos.O receptor de NMDA (NMDAR) é um receptor ionotrópico com o qual o A2AR pode interagir. Deste modo, dada a particular importância do NMDAR para o fenómeno de neurotransmissão no hippocampo, este trabalho explora de que forma é que os A2AR modulam as correntes dependentes de NMDAR nas sinapses entre os neurónios piramidais do CA1 e as colaterais de Schaffer (SC) no hipocampo.Mostrou-se que a activação exógena do A2AR com o seu agonista selectivo CGS21680 diminui as correntes excitatórias pós-sinápticas evocadas (eEPSC) dependentes do receptor de NMDA, um efeito que deixa de ser observado na presença do antagonista selectivo dos A2AR, SCH58261. Além disso, SCH58261 diminui a amplitude dos eEPSCs em condições basais o que sugere que os A2AR poderão exercer um controlo tónico destas correntes nos neurónios piramidais do CA1. Finalmente, com este trabalho observou-se ainda, que as preparações in vitro utilizadas têm uma grande quantidade de adenosina endógena. A sua remoção, porém, não previne o efeito induzido pelo antagonista selectivo dos A2AR, continuando a observar-se uma diminuição na amplitude de eEPSC dependentes de NMDAR nas sinapses CA1-SC.A principal hipótese para explicar esta aparente contradição de resultados tem a ver com a possível existência de duas sub-populações de A2AR que podem afectar as correntes dependentes de NMDAR de maneira oposta. Contudo, o estudo de qual a população de A2AR que é responsável por cada um dos efeitos, assim como quais os mecanismos por detrás desta evidente modulação dos A2AR sobre os NMDAR permanece por clarificsr, o que poderá servir de basede trabalho futuro.
Outro - 016684 - PTDC/NEU-NMC/4154/2014 (POCI-01-0145-FEDER-016684) - Papel dos astrócitos no controlo da memória- foco nos recetores adenosina A2A - Fundação para a Ciência e a Tecnologia
Outro - COMPETE POCI-01-0145-FEDER-007440 - Trabalho multidisciplinar no âmbito da neurociência cognitiva na saúde e na doença - Fundação para a Ciência e a Tecnologia

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
Recetores acoplados à proteína G são alvos terapêuticos-chave para muitas condições patológicas. Estudos demonstraram que os recetores A2A de adenosina (A2AR) e D2 de dopamina (D2R) acoplados à proteína G formam heterómeros A2AR-D2R no estriado. A estequiometria deste heterómero encontra-se alterada na doença de Parkinson (DP) e a sinalização mediada pelos recetores A2A pode ser promovida. Deste modo, os recetores A2A são o alvo farmacológico de eleição na DP que mais tem recebido atenção nos últimos anos. No entanto, a ubiquidade deste recetor tem dificultado a seletividade no tempo e no espaço de fármacos baseados em adenosina, levando à diminuição do seu efeito terapêutico. A fotofarmacolgia tem vindo a desenvolver novos fármacos, por exemplo, compostos caged nos quais a sua atividade pode ser controlada de uma forma espácio-temporal através do uso de uma fonte de luz. MRS7145 (caged-SCH442416) foi o primeiro antagonista caged dos recetores A2A a ser sintetizado. Quando irradiado (405 nm), através de uma fonte externa de luz, este composto exibiu um perfil de antagonista relativamente aos recetores A2A em células vivas. No modelo de murganho da DP, induzido pela injeção de 6 hidroxidopamina (6-OHDA), demonstrou melhorias dos sintomas motores causados por esta doença. No entanto, a aplicação deste método envolve uma cirurgia ao cérebro complicada e requer o implante de fibras óticas, o que pode limitar a sua utilidade. O objetivo da presente tese foi avaliar se a bioluminescência gerada pela oxidação de coelenterazina 400a, através da enzima nanoluciferase (NL) acoplada ao recetor A2A (A2ARNL), seria suficiente para efetuar a libertação (uncaging) do composto MRS7145 em células vivas. Inicialmente, foi criada uma linha celular estável de células HEK-293 expressando permanentemente A2ARNL. Foram detetados baixos níveis de expressão deste recetor na membrana de células pertencentes à linha celular estável, comparativamente aos níveis detetados em células transientemente transfectadas com o mesmo vetor. Todavia, os valores acumulados de monofosfato cíclico de adenosina (cAMP) e alterações em impedância celular obtidos, após incubação com forskolina (ativador da adenilato ciclase), CGS21680 (agonista) e ZM241385 (antagonista), asseguraram a funcionalidade do recetor na linha celular criada. Além disso, 15 minutos de incubação com coelenterazina 400a, ou com o seu solvente, etanol, não provocou qualquer diminuição na viabilidade celular. No entanto, incubação com coelenterazina 400a levou à diminuição dos níveis de cAMP produzidos pela ação do agonista CGS21680, alterando as condições da ativação do recetor. Coelenterazina 400a não teve qualquer efeito na atividade do composto SCH442416 em bloquear o recetor A2A. Por fim, foi avaliado o bloqueio autónomo do recetor A2A mediado pela libertação (uncaging) do composto MRS7145 dependente da bioluminescência gerada pela enzima nanoluciferase acoplada. Quando incubado com coelenterazina 400a, o composto MRS7145 impediu a ativação do recetor de A2A mediada pelo agonista CGS21680. Resumindo, o método descrito baseado em bioluminescência, demonstrou a primeira evidência do uncaging do composto MRS7145 e subsequente libertação do composto SCH442416 em células vivas, sob um controlo espácio-temporal e independente de uma fonte externa de luz. Este método apresenta grande potencial para ser futuramente otimizado e aplicado no tratamento de distúrbios motores, incluindo a doença de Parkinson e, ainda, em terapias que visem a utilização de fármacos cuja atividade possa ser controlada por uma fonte de luz.
G protein-coupled receptors are key therapeutic targets for many pathological conditions. Studies support that the G protein-coupled adenosine A2A receptor (A2AR) and dopamine D2 receptor (D2R) form A2AR‐D2R heteromers in the striatum. The stoichiometry of this receptor heteromer is altered in Parkinson’s disease (PD) and A2AR signaling may be promoted. Hence, the A2AR represents the latest pharmacological target in PD. However, owing to the ubiquity of this receptor, it has been difficult to achieve drug selectivity in time and space for adenosine-based drugs which diminishes their therapeutic use. Photopharmacology has been developing novel drugs, e.g., caged-compounds, whose activity can be controlled in a spatiotemporal-manner with the use of a light source. MRS7145 (caged-SCH442416) was the first A2AR photo-caged antagonist to be synthesized whose external irradiation (405 nm) showed a light-dependent A2AR antagonist activity in living cells and antiparkinsonian effects in a hemiparkinsonian 6-hyroxydopamine (6-OHDA)-lesioned mouse model. However, this approach involves laborious brain surgery and optic fiber implants that may limit its utility. The aim of the present thesis was to evaluate whether the bioluminescence produced by an A2AR-coupled nanoluciferase (NL)-mediated coelenterazine 400a oxidation would be able to uncage MRS7145 in living cells. To begin with, a HEK-293 stable cell line expressing a previously engineered A2ARNL was created. Low receptor expression levels were found at the membrane of cells belonging to the stable cell line when compared to transiently transfected cells with the same construct. However, the cAMP accumulated levels and changes in cellular impedance obtained upon cell incubation with forskolin (adenylyl cyclase stimulator), CGS21680 (agonist), and ZM241385 (antagonist), ensured receptor functionality. Moreover, 15 minutes incubation with coelenterazine 400a or its solvent, ethanol, had no effect in decreasing cell viability, while incubation with coelenterazine 400a decreased CGS21680-induced cAMP accumulation altering receptor activation in the generated stable cell line. Coelenterazine 400a incubation did not affect SCH442416-induced receptor blockade. Finally, the autaptic A2AR blockade mediated by receptor’s bioluminescence-dependent uncaging of MRS7145 was evaluated. MRS7145 precluded CGS21680-induced receptor activation when incubated with coelenterazine 400a in living cells. Altogether, the described bioluminescence-based method provided the first proof of concept in uncaging MRS7145 and subsequent photorelease of SCH442416 in living cells, under a spatiotemporal control and independently of an external light source. This method demonstrates potential to be further optimized to be applied in the management of movement disorders, including Parkinson’s disease, and other prospective smart therapies which aim to utilize photocontrolled drugs.
Outro - SAF2017‐87349‐R from Ministerio de Economía y Competitividad‐Spanish Government, I+D (Project title: “Lighthing up dopamine, adenosine and GPR37 receptors in neurological and neuropsychiatric diseases”).

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012
O hipocampo é a estrutura cerebral mais estudada na investigação neurocientífica. A subdivisão CA1 do hipocampo contém a população neuronal que apresenta menor heterogeneidade entre neurónios, encontrando-se organizada em camadas bem definidas, o que torna os neurónios fáceis de identificar e de estudar (Szilágyi et al. 2011). O neurónio é a unidade sinalizadora individual do sistema nervoso central (SNC). Os neurónios estão inseridos numa complexa rede de circuitos neuronais que se encontra distribuída por todo o cérebro, comunicando entre si através de sinais eléctricos designados como potenciais de acção e potenciais sinápticos (Adrian, 1957). A transmissão de informação entre um neurónio localizado pré-sinapticamente e uma célula pós-sináptica designa-se como transmissão sináptica (Breathnach, 2005). As sinapses químicas envolvem a libertação pré sináptica de neurotransmissores para a fenda sináptica (Connors & Long, 2004), activando receptores específicos localizados na membrana da célula pós-sináptica (Schoepp et al. 1999). Os receptores NMDA (N-methyl-D-aspartate) são receptores ionotrópicos cujo principal agonista endógeno é o glutamato (Bakkar et al. 2011. As suas principais características são a dependência de voltagem, elevada permeabilidade ao cálcio e cinética de activação/desactivação lenta (Doherty & Sladek, 2011). Os receptores NMDA são moléculas heteroméricas formadas pelas subunidades GluN1, GluN2 e GluN3 (Groc et al. 2009). Na região CA1 do hipocampo, são tetra heteromeros compostos por duas subunidades GluN1 e duas subunidades GluN2 (Bakkar et al. 2011), podendo classificar-se como sinápticos ou extrasinápticos (Groc et al. 2009). A actividade dos receptores NMDA está associada, de forma paradoxal, a fenómenos indutores de sobrevivência neuronal e de plasticidade sináptica (Larkman & Jack, 1995; Lipton & Nakanishi, 1999), e a episódios neurodegenerativos associados a morte neuronal (Xu et al. 2009). A ambivalência nas consequências da actividade dos receptores NMDA recebe o nome de “paradoxo dos receptores NMDA” (Hardingham & Bading, 2010), sendo atribuída à localização sináptica destes receptores. De facto, a actividade de receptores NMDA sinápticos aparenta estar associada a fenómenos de protecção neuronal, ao passo que a actividade de receptores NMDA extrasinápticos conduz a fenómenos de apoptose neuronal (Hardingham & Bading, 2010; Stark & Bazan, 2011). A actividade de receptores NMDA pode ser influenciada pela acção de neuromoduladores do SNC. A neuromodulação consiste na habilidade neuronal para modificar as propriedades eléctricas dos neurónios em resposta a mudanças bioquímicas intracelulares resultantes de estímulos sinápticos ou hormonais, permitindo ao SNC adaptar a sua capacidade de controlar funções fisiológicas num ambiente em constante mudança (Kaczmarek & Levitan, 1987). A adenosina é um neuromodulador que medeia os seus efeitos biológicos através de quatros subtipos de receptores: receptores A1, A2A, A2B e A3 (A1Rs, A2ARs, A2BRs eA3Rs) (Tsutsui et al 2004). Grande parte das acções neuromoduladoras da adenosina são mediadas pelos receptores A1 e A2A (Gomes et al. 2010). Os receptores A1 ligam-se a proteínas Gi, cuja actividade inibe a actividade da adenilato ciclase (van Calker et al. 1979). Por seu turno, os receptores A2A ligam-se a proteínas Gs, que estimulam a expressão de adenilato ciclase (Corvol et al. 2001). Enquanto os receptores A1 se encontram amplamente disseminado pelo cérebro (Dunwiddie & Masino, 2001), os receptores A2A estão distribuídos em áreas especificas como o estriado (Haas & Selbach, 2000), embora estejam também presentes em outras áreas cerebrais (Fredholm et al. 2005). A adenosina é uma substância neuromoduladora capaz de inibir ou de estimular a neurotransmissão no SNC (Fredholm & Dunwiddie, 1988). Os receptores A1 reduzem a libertação de neurotransmissor e inibem a transmissão sináptica. As acções inibitórias mais proeminentes dos receptores A1 estão identificadas em sistemas de transmissão glutamatérgica excitatória, através da inibição na libertação do neurotransmissor glutamato (Barrie & Nicholls, 1993). Apesar da actividade dos receptores A2 ter um impacto limitado no controlo da transmissão sináptica basal, eles são cruciais no controlo da plasticidade sináptica (Gomes et al. 2010). No hipocampo, a utilização de agonistas (Cunha et al. 1996) e antagonistas (Li & Henry, 1998) específicos para estes receptores provocam, respectivamente, aumentos e reduções na transmissão sináptica excitatória. A nível pré-sináptico, os receptores A2A desempenham um papel facilitatório na libertação de glutamato (Lopes et al. 1999). No hipocampo, os efeitos moduladores dos receptores A2A sobre a actividade dos receptores NMDA é menos bem conhecida. Noutras estruturas cerebrais há evidências de que, a nível pós-sináptico, os receptores A2A modulam a actividade dos receptores NMDA (Wirkner et al. 2004; Tebano et al. 2005; Rebola et al. 2008). De igual forma, sabe-se que em neurónios CA1 piramidais, os receptores A2A de adenosina controlam de forma directa a activação de receptores AMPA de glutamato (Dias et al. 2002). Este trabalho foi desenvolvido com o objectivo de compreender se a activação farmacológica de receptores A2A de adenosina pode modular a actividade de receptores NMDA na região CA1 do hipocampo. Utilizando fatias de hipocampo obtidas a partir de ratos Wistar jovem-adultos, os dados foram adquiridos através de técnicas de electrofisiologia, nomeadamente, procedimentos de patch-clamp utilizando a configuração whole-cell. Primeiramente, foi necessário garantir que as correntes evocadas refletiam exclusivamente actividade de receptores NMDA. Assim sendo, foi utilizado CNQX (10μM), um antagonista competitivo selectivo para receptores de glutamato AMPA e kainato. Os resultados não demonstram alterações significativas nas correntes (99%±2.9% n=3, p>0.05), o que permite concluir que as correntes registadas estavam apenas a ser evocadas pela actividade dos receptores NMDA. De igual forma, para garantir que este efeito está dependente da activação de receptores NMDA, foi utilizado um antagonista especifico destes receptores. A aplicação de DL-APV (50μM) provocou um decréscimo muito significativo nas correntes registadas (76%±4.9% n=3, p<0.005). De seguida, com o objectivo de tentar encontrar um efeito modulador dos receptores A2A sobre a actividade dos receptores NMDA, começou-se por avaliar os efeitos do CGS 21680, um agonista selectivo dos receptores A2A, sobre a actividade dos receptores NMDA. A adição de CGS 21680 (30nM) provocou um aumento significativo nas correntes mediadas pela actividade dos receptores NMDA (23%±4,7% n=6, p<0.005). Estes dados apontam para um efeito modulador dos receptores A2A que potencia a actividade dos receptores NMDA em células CA1 do hipocampo. Para assegurar que este efeito reflecte um efeito modulador dos receptores A2A, foi adicionado o agonista CGS 21680 na presença prévia do antagonista selectivo dos receptores A2A, o SCH 58261. A adição de SCH 58261 (100nM) não causou diferenças significativas na amplitude das correntes (3%±14,5% n=3, p>0.05), o que significa que os receptores A2A endógenos não contribuem para o efeito anteriormente registado. A adição subsequente do agonista CGS 21680 (30nM), após a adição prévia do antagonista durante 10 minutos, não provocou alterações significativas na amplitude das correntes registadas (2%±9,0% n=5, p>0.05). O facto do efeito potenciador do agonista ser prevenido pela presença do antagonista dos receptores A2A, permite concluir que este efeito modulador é mediado por receptores A2A. Estes resultados sugerem que os receptores A2A modulam a actividade dos receptores NMDA, resultando numa potenciação das correntes pós-sinápticas mediadas pelos receptores NMDA. Estes resultados reflectem um efeito ainda não descrito das capacidades neuromoduladoras dos receptores A2A. Futuramente, seria importante discriminar se as correntes mediadas reflectem a actividade de receptores NMDA localizados sinapticamente ou extrasinapticamente, uma vez que a actividade de receptores NMDA localizados em diferentes áreas sinápticas tem implicações diferentes para a saúde neuronal. A memantina, um fármaco que bloqueia preferencialmente a actividade de receptores NMDA extrasinápticos, poder-se-á revelar uma ferramenta útil para uma investigação futura sobre o tema.
Hippocampal excitatory synaptic plasticity is often considered the synaptic basis for memory formation. NMDAR activity is deeply involved in long-lasting changes in synaptic plasticity. Adenosine modulatory actions upon excitatory glutamatergic transmission are well described. However, the modulatory actions of adenosine A2ARs upon NMDARs activity in CA1 pyramidal cells have never been reported. Thus, the effect of A2AR activation on NMDARs-mediated postsynaptic currents (PSCs) was examined in CA1 pyramidal neurons of young (3-10 weeks) rat hippocampal slices, by using the whole-cell patch-clamp technique (Vh = -60mV). NMDARs-mediated currents were evoked through pressure application of NMDA (150μM) (selective NMDAR agonist) directly onto the cell soma. Bath application of A2AR agonist CGS 21680 (30nM) induced significant increases on NMDA-evoked currents (23%±4,7% n=6, p<0.005). To further address if this effect was caused by an A2AR-mediated modulation of NMDA-evoked PSCs, CGS 21680 (30nM) was superfused in the presence of SCH 58261 (100nM), a selective A2AR antagonist, which was added to the perfusion 10 minutes before the agonist. NMDA-evoked PSCs were not significantly altered by the presence of CGS 21680 (30nM) when A2ARs were previously blocked by SCH 58261 (100nM) (2%±9,0% n=5, p>0.05). To assure that the measured currents were elicited by NMDARs activity, CNQX (10μM) was used to block the activity of AMPA/kainate receptors. The results show no significant changes in NMDA-evoked PSCs in the presence of CNQX (99%±2.9% n=3, p>0.05), suggesting that these currents were mediated by NMDARs. Finally, the use of DL-APV (50μM) - selective NMDARs antagonist - served to further assure that NMDARs mediated the observed currents. In the presence of DL-APV, NMDAevoked postsynaptic currents significantly decreased (76%±4.9% n=3, p<0.005). Together these results allow to conclude that A2ARs exert a modulatory effect over NMDARs activity at the CA1 neurons of the hippocampus, resulting in potentiation of NMDA-evoked PSCs.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Magdaléna Svobodová Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Assoc. Prof. Maria da Glória Correia da Silva Queiroz, Ph.D. Title of diploma thesis: Microglia control adenosine A2A-receptor mediated astrogliosis In the central nervous system, astrocytes and microglia are the main cells coordinating the inflammatory response. During inflammation, dying or temporarily damaged cells release ATP, as a danger-associated signal molecule, that contributes to the induction of astrogliosis and promotes clearance of the debris by immune cells such as microglia. Adenosine that results from ATP metabolism also stimulates astrogliosis. However, the effects of adenosine on astrogliosis may be more complex, since it also modulates microglia phenotype and microglia have been shown to prevent excessive astroglial proliferation mediated by nucleotides. In this context, ATP and adenosine are assumed as relevant signalling molecules in the control of astrogliosis and its modulation by microglia. However, it is still unknown whether and how microglia modulate adenosine-mediated astrogliosis. The present study aims to clarify the impact of microglia in the control of adenosine-induced astrogliosis. Two...

Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de Coimbra
Adenosine is a prototypic modulator of synaptic transmission in the CNS; it mainly controls excitatory transmission through a coordinated action of inhibitory A1 receptors and facilitatory adenosine A2A receptors (A1R, A2AR) and, albeit their physiological and pathological role have mainly been assumed to result from their direct action on neurons, they are also present in astrocytes where they control astrogliosis, proliferation, cell death, and the release of neurotrophic factors and interleukins. Astrocytes are the dominant subclass of non-neuronal glial cells of the brain; they are physically associated with synapses prompting the tripartite synapse concept to highlight their relevance in integrating neuronal networks by reciprocal chemical signaling. Astrocytes format synaptic plasticity since they are responsible for clearance of extracellular glutamate through glutamate transporters. This highlights the crucial involvement of astrocytes on the abnormal glutamate over-excitation implicated in both acute CNS injuries and diverse chronic neurodegenerative disorders. The group of Purines at CNC recently showed that astrocytic glutamate uptake is diminished upon activation of astrocytic A2AR, both under physiological and pathological conditions, which supports the ability of astrocytic A2AR to control pathophysiological processes involving the activity of glutamate transporters. Additionally, A2AR are aberrantly up-regulated upon different brain insults. Therefore, we now prompted the hypothesis of blocking astrocytic A2AR as a novel and promising strategy to prevent abnormal glutamate over-excitation, thus preventing biochemical-, functional- and behavioural-associated modifications. Hence, we sought to validate novel tools for selectively and region-specific down-regulate astrocytic A2AR to further probe their efficacy under physiological and pathological conditions. This work was organized in 2 main steps: i) we first incorporated into a lentivector coated with Mokola Lyssavirus G glycoprotein (Mok-G) an RNA interference strategy to down-regulate A2AR also carrying a reporter gene, enhanced green fluorescent protein (EGFP) to allow cells to be identifiable,; ii) we then tested whether Mok-G coated lentivirus were able to selectively and efficiently transduce astrocytes in vitro (primary astrocyte cell cultures) and in vivo (into selected brain regions such as prefrontal cortex, striatum and hippocampus) for delivering shA2AR constructs to downregulate A2AR expression and density. We evaluated viral spreading and cell-type transduction through immunofluorescent co-localization of the reporter gene EGFP with glial (GFAP and vimentin) and neuronal (NeuN) markers. The present study showed that Mokola-G-coated lentivirus encoding shA2AR successfully infects astrocytes and down-regulate astrocytic A2A receptors expression and density, at least in vitro. Major concerns should be considered when it comes to their in vivo application, especially since there are different transduction efficiencies as well as selectivity of astrocytic targeting in the adult rodent brain with further implications for therapeutic gene transfer.
Adenosina é um modulador típico da transmissão sináptica no sistema nervoso central (SNC); controla maioritariamente a transmissão excitatória através da acção coordenada dos receptores inibitórios A1 e excitatórios A2A e, apesar do seu papel fisiológico e patológico resultar da sua ação preponderantemente em neurónios, estão também presentes em astrócitos onde controlam a astrogliose, a proliferação, a morte celular, e a libertação de factores neurotróficos e interleucinas. Os astrócitos são as células da glia não neuronais mais prevalentes no cérebro e estão fisicamente associados às sinapses onde a reciprocidade da sinalização química permite o funcionamento íntegro dos circuitos neuronais, o que originou o conceito de sinapse tripartida. Os astrócitos são importantes mediadores da plasticidade sináptica uma vez que eliminam o glutamato extracelular através de transportadores de glutamato. Este facto reveste-se de particular relevo em situações de sobre-excitação anormal pelo glutamato caraterístico de lesões agudas no SNC bem como em diversas doenças neurodegenerativas. O grupo de Purinas demonstrou recentemente que a activação dos receptores A2A astrocitários diminui a captação de glutamato em condições fisiológicas e patológicas o que atesta capacidade dos receptores A2A astrocitários em controlar processos patológicos através da actividade dos transportadores de glutamato. Uma vez que a densidade de receptores A2A (A2AR) está anormalmente aumentada em diversas situações de dano cerebral, propomos agora a hipótese de que o bloqueio dos receptores A2A astrocitários se constitua como uma nova estratégia promissora para prevenir a sobre-excitação anormal do glutamato prevenindo assim as modificações bioquímicas, funcionais e comportamentais associadas. Por essa razão, validámos novas ferramentas para diminuir local e seletivamente a expressão de receptores A2A astrocitários e posteriormente comprovar a sua eficácia em condições fisiológicas e patológicas. Este trabalho foi executado em duas etapas: i) incorporou-se em um vector viral revestido com a glicoproteína G do vírus Mokola um RNA de interferência (shRNA) para diminuir a expressão astrocitária de A2AR e também um gene repórter, a proteína fluorescente verde (EGFP) para permitir a identificação das células infectadas; ii) testouse a eficiência de infeção do lentivírus e a sua selectividade para astrócitos quer in vitro (em culturas primárias) quer in vivo (por administração dos lentivírus em regiões particulares do cérebro, tais como o córtex pré-frontal, o estriado e o hipocampo) e avaliou-se a diminuição da densidade dos receptores A2A astrocitários mediada pelos shRNAs. Avaliou-se a difusão do vírus bem como a transdução celular através da colocalização por imunofluorescência do EGFP com marcadores de astrócitos (GFAP e vimentina) e de neurónios (NeuN). Este estudo demonstrou que o lentivirus revestido com a glicoproteína G do vírus Mokola contendo shA2AR é eficiente na infeção de astrócitos bem como na diminuição da densidade de A2AR in vitro. Uma vez que a eficiência de transdução bem como a seletividade para astrócitos destes vírus parece ser distinta em diferentes regiões do cérebro de roedor com implicações para a sua aplicabilidade terapêutica, a utilização desta estratégia deve ser ponderada.

碩士
國立陽明大學
神經科學研究所
89
The rat A2A adenosine receptor (A2A-R) is heavily enriched in the striatum and is also expressed at relatively low levels in other areas of the brain. Gene of A2A-R contains two independent promoters (P1 and P2). Transcripts produced from these two promoters differ in the length of their 5'-untranslated regions (5'-UTR), but encode the same protein. To further characterize the transcriptional regulation of the A2A-R gene in vivo, a fusion gene consisting the 4.8 kb 5'-flanking region / full length 5'UTR of the A2A-R gene and the coding region of the N-lacZ (n-β-galactosidase) was created to produce mice harboring the indicated fusion gene. Enzymatic analysis and histochemistry determinations reveal that the transgene (A2A-R promoter /β-galactosidase) was expressed mainly in the brain, indicating that the 4.8 kb 5'-flanking region / 5'UTR of the A2A-R gene might contain important elements for its expression in the brain. Furthermore, using the immunohistochemical double staining technique, I found that the transgene colocalized with the endogenous mouse A2A-R in various regions of the brain. Some of the transgene-positive cells are neurons and some are glial cells. Colocalization analysis of the transgene and various markers in the striatum shows that this transgene is located in both cholinergic and striatopallidal neurons (GABAergic neurons) as predicted. Nevertheless, the transgene-positive cells in the striatum where A2A-R is enriched only contributed to a small portion of the overall A2A-R-positive cells. To ascertain that the transgenic construct we designed contains the functional A2A-R promoters for its expression in the striatum, 5'-RACE experiments were carried out to evaluate the major functional A2A-R promoter in the striatum. To our surprise, results of 5'RACE suggest that additional exons and introns might exist for the rat A2A-R gene. Further analysis is required to clarify the complete gene structure of the rat A2A-R gene.