Esse estudo foi realizado para verificar se a exposição a separações repetidas (por diferentes intervalos de tempo) de mães dos seus filhotes no período neonatal poderiam ser classificadas como indutoras de um estado do tipo deprimido em genitoras. Sessenta ratas Wistar prenhes foram divididas em 3 grupos: controle, separação por 10 minutos e separação por 3 horas. As intervenções neonatais foram realizadas nos dias 1-10 pós parto. Após o desmame as genitoras foram submetidas ao teste do nado forçado, ao teste do labirinto em cruz elevado e ao teste do odor de predador. Também foi avaliado o comportamento alimentar e os padrões de reatividade a um sabor doce e a um sabor amargo. Foi medido os níveis de ocitocina no líquido cefaloraquidiano, corticosterona plasmática e atividade hipocampal Na+, K+-ATPase, assim como a atividade das enzimas antioxidantes catalase, glutationa peroxidase, superoxido dismutase, produção de radicais livres, e a produção de óxido nítrico, além dos níveis dos receptores A2A de adenosina e D2 de dopamina no estriado dorsoventral e no hipocampo. Foi observado que somente a separação por 3 h induziu um aumento significativo no tempo de imobilidade no teste do nado forçado, o que é consistente com estudos prévios. A atividade hipocampal da Na+, K+-ATPase se mostrou diminuída no grupo separado por 10 minutos e mais significativamente diminuída nas genitoras submetidas a separação por 3 horas de seus filhotes. Adicionalmente, os níveis de ocitocina no líquido cefaloraquidiano se encontravam aumentados no grupo separado por 10 minutos, o que pode estar relacionado a um aumento no cuidado materno, induzido por esta manipulação dos filhotes, por parte das genitoras, como reportado na literatura. Uma redução nos níveis de óxido nítrico no hipocampo das genitoras separadas por 3 horas foi observado Nessas genitoras também foi verificado um aumento no comportamento de risco, uma diminuição no sentimento prazeroso frente a uma solução doce e aumento na sensibilidade a uma solução aversiva, o que é congruente a um perfil de estado do tipo deprimido. Além disso, nós verificamos uma diminuição na quantidade do receptor de dopamina D2 no estriado das mães submetidas a separação por 3 horas dos filhotes, o que poderia ser relacionado com uma diminuição no prazer (anedonia) que acontece na depressão. Conclui-se que a retirada dos filhotes das mães por longos períodos tornam essas mães mais susceptíveis ao desenvolvimento de características depressivas. / This study was carried out to ascertain whether exposure to repeated separations (different times) of mothers from their pups in the neonatal period could be classified as an induction of a depressive-like state in dams. Sixty Wistar rats were divided into 3 groups: control, brief separation and long-term separation. The neonatal interventions were done on postpartum days 1-10. After weaning, the dams were subjected to the forced swimming test, elevated plus maze and predator odor test. It was also evaluated the feeding behavior and the taste reactivity patterns to a sweet and to a bitter solution. It was mesaured cerebral spinal fluid oxytocin, plasma corticosterone, and hippocampal Na+, K+-ATPase activity, as well as the activity of the antioxidant enzymes catalase, glutathione peroxidase, superoxide dismutase, free radicals production, and the production of nitric oxide and the levels of adenosine A2A and dopamine D2 receptors in the dorsoventral striatum and hippocampus. It was observed that only the 3 h separation induced a significant increase in the immobility time of rats in the forced swimming test, which is consistent with previous studies. Hippocampal Na+, K+-ATPase activity was decreased in the brief separated group and more significantly decreased in dams subjected to 3h separation from their pups. Additionally, cerebral spinal fluid oxytocin was increased in dams of the brief separated group, which may be related to the increased handled-induced maternal care, as reported in the literature. A reduction in nitric oxide levels in the hippocampus in dams of the long separated group was also observed. It was also verify an increase in the risk-taking behavior by the 3h separated mothers. The 3h separated mother also demonstrated a diminished feeling of pleasure with a sucrose solution and a increased sensibility to a aversive solution, wich is congruent with a depressive like state profile. Furthermore, we shown a decrease in the dopamine D2 receptor quantity in the striatum of the 3 h separated mothers, wich could be related to a decrease in pleasure feeling (anhedonia) experienced in depression. It is concluded that the withdrawal of pups from their mothers for long periods make the mothers more susceptible to the development of depressive features.

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.

Esse estudo foi realizado para verificar se a exposição a separações repetidas (por diferentes intervalos de tempo) de mães dos seus filhotes no período neonatal poderiam ser classificadas como indutoras de um estado do tipo deprimido em genitoras. Sessenta ratas Wistar prenhes foram divididas em 3 grupos: controle, separação por 10 minutos e separação por 3 horas. As intervenções neonatais foram realizadas nos dias 1-10 pós parto. Após o desmame as genitoras foram submetidas ao teste do nado forçado, ao teste do labirinto em cruz elevado e ao teste do odor de predador. Também foi avaliado o comportamento alimentar e os padrões de reatividade a um sabor doce e a um sabor amargo. Foi medido os níveis de ocitocina no líquido cefaloraquidiano, corticosterona plasmática e atividade hipocampal Na+, K+-ATPase, assim como a atividade das enzimas antioxidantes catalase, glutationa peroxidase, superoxido dismutase, produção de radicais livres, e a produção de óxido nítrico, além dos níveis dos receptores A2A de adenosina e D2 de dopamina no estriado dorsoventral e no hipocampo. Foi observado que somente a separação por 3 h induziu um aumento significativo no tempo de imobilidade no teste do nado forçado, o que é consistente com estudos prévios. A atividade hipocampal da Na+, K+-ATPase se mostrou diminuída no grupo separado por 10 minutos e mais significativamente diminuída nas genitoras submetidas a separação por 3 horas de seus filhotes. Adicionalmente, os níveis de ocitocina no líquido cefaloraquidiano se encontravam aumentados no grupo separado por 10 minutos, o que pode estar relacionado a um aumento no cuidado materno, induzido por esta manipulação dos filhotes, por parte das genitoras, como reportado na literatura. Uma redução nos níveis de óxido nítrico no hipocampo das genitoras separadas por 3 horas foi observado Nessas genitoras também foi verificado um aumento no comportamento de risco, uma diminuição no sentimento prazeroso frente a uma solução doce e aumento na sensibilidade a uma solução aversiva, o que é congruente a um perfil de estado do tipo deprimido. Além disso, nós verificamos uma diminuição na quantidade do receptor de dopamina D2 no estriado das mães submetidas a separação por 3 horas dos filhotes, o que poderia ser relacionado com uma diminuição no prazer (anedonia) que acontece na depressão. Conclui-se que a retirada dos filhotes das mães por longos períodos tornam essas mães mais susceptíveis ao desenvolvimento de características depressivas. / This study was carried out to ascertain whether exposure to repeated separations (different times) of mothers from their pups in the neonatal period could be classified as an induction of a depressive-like state in dams. Sixty Wistar rats were divided into 3 groups: control, brief separation and long-term separation. The neonatal interventions were done on postpartum days 1-10. After weaning, the dams were subjected to the forced swimming test, elevated plus maze and predator odor test. It was also evaluated the feeding behavior and the taste reactivity patterns to a sweet and to a bitter solution. It was mesaured cerebral spinal fluid oxytocin, plasma corticosterone, and hippocampal Na+, K+-ATPase activity, as well as the activity of the antioxidant enzymes catalase, glutathione peroxidase, superoxide dismutase, free radicals production, and the production of nitric oxide and the levels of adenosine A2A and dopamine D2 receptors in the dorsoventral striatum and hippocampus. It was observed that only the 3 h separation induced a significant increase in the immobility time of rats in the forced swimming test, which is consistent with previous studies. Hippocampal Na+, K+-ATPase activity was decreased in the brief separated group and more significantly decreased in dams subjected to 3h separation from their pups. Additionally, cerebral spinal fluid oxytocin was increased in dams of the brief separated group, which may be related to the increased handled-induced maternal care, as reported in the literature. A reduction in nitric oxide levels in the hippocampus in dams of the long separated group was also observed. It was also verify an increase in the risk-taking behavior by the 3h separated mothers. The 3h separated mother also demonstrated a diminished feeling of pleasure with a sucrose solution and a increased sensibility to a aversive solution, wich is congruent with a depressive like state profile. Furthermore, we shown a decrease in the dopamine D2 receptor quantity in the striatum of the 3 h separated mothers, wich could be related to a decrease in pleasure feeling (anhedonia) experienced in depression. It is concluded that the withdrawal of pups from their mothers for long periods make the mothers more susceptible to the development of depressive features.

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterised by a reduction of dopamine concentration in the striatum due to degeneration of dopaminergic neurons in the substantia nigra. Currently, first line treatment of PD includes the use of dopamine precursors, dopamine agonists and inhibitors of enzymatic degradation of dopamine, in an effort to restore dopamine levels and/or its effects. However, all these therapeutic strategies are only symptomatic and unfortunately do not slow, stop or reverse the progression of PD. From the discovery of adenosine A2A receptor-dopamine D2 receptor heteromers and the antagonistic interaction between these receptors, the basis of a new therapeutic approach towards the treatment of PD emerged. Adenosine A2A receptor antagonists have been shown to decrease the motor symptoms associated with PD, and are also potentially neuroprotective. The possibility thus exists that the administration of an adenosine A2A antagonist may prevent further neurodegeneration. Furthermore, the antagonism of adenosine A1 receptors has the potential of treating cognitive deficits such as those associated with Alzheimer's disease and PD. Therefore, dual antagonism of adenosine A1 and A2A receptors would be of great benefit since this would potentially treat both the motor as well as the cognitive impairment associated with PD. The affinities (Ki-values between 0.6 mM and 38 mM) of a series of 1,4-dihydropyridine derivatives were previously illustrated for the adenosine A1, A2A and A3 receptor subtypes by Van Rhee and co-workers (1996). These results prompted this pilot study, which aimed to investigate the potential of the structurally related 3,4-dihydropyrimidin-2(1H)-ones (dihydropyrimidones) and 2-amino-1,4-dihydropyrimidines as adenosine A1 and A2A antagonists. In this pilot study, a series of 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines were synthesised and evaluated as adenosine A1 and A2A antagonists. Since several adenosine A2A antagonists also exhibit MAO inhibitory activity, the MAO-inhibitory activity of selected derivatives was also assessed. A modified Biginelli one pot synthesis was used for the preparation of both series of compounds under solvent free conditions. A mixture of a β- diketone, aldehyde and urea/guanidine hydrochloride was heated for an appropriate time to afford the desired compounds in good yields. MAO-B inhibition studies comprised of a fluorometric assay where kynuramine was used as substrate. A radioligand binding protocol described in literature was employed to investigate the binding of the compounds to the adenosine A2A and A1 receptors. The displacement of N-[3H]ethyladenosin-5’-uronamide ([3H]NECA) from rat striatal membranes and 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat whole brain membranes, was used in the determination of A2A and A1 affinity, respectively. The results showed that both 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines had weak adenosine A2A affinity, with the p-fluorophenyl substituted dihydropyrimidone derivative (1h) in series 1, exhibiting the highest affinity for the adenosine A2A receptor (28.7 μM), followed by the p-chlorophenyl dihydropyrimidine derivative (2c) in series 2 (38.59 μM). Both series showed more promising adenosine A1 receptor affinity in the low micromolar range. The p-bromophenyl substituted derivatives in both series showed the best affinity for the adenosine A1 receptor with Ki-values of 7.39 μM (1b) and 7.9 μM (2b). The pmethoxyphenyl dihydropyrimidone (1d) and p-methylpneyl dihydropyrimidine (2e) derivatives also exhibited reasonable affinity for the adenosine A1 receptor with Ki-values of 8.53 μM and 9.67 μM, respectively. Neither the 3,4-dihydropyrimidones nor the 2-amino-1,4- dihydropyrimidines showed MAO-B inhibitory activity. Comparison of the adenosine A2A affinity of the most potent derivative (1h, Ki = 28.7 μM) from this study with that of the previously synthesised dihydropyridine derivatives (Van Rhee et al., 1996, most potent compound had a Ki = 2.74 mM) reveals that an approximate 100- fold increase in binding affinity for A2A receptors occurred. However, KW6002, a known A2A antagonist, that has already reached clinical trials, has a Ki-value of 7.49 nM. The same trend was observed for adenosine A1 affinity, where the most potent compound (1b) of this study exhibited a Ki-value of 7.39 μM compared to 2.75 mM determined for the most potent dihydropyridine derivatives (Van Rhee et al., 1996). N6-cyclopentyladenosine (CPA), a known adenosine A1 agonist that was used as a reference compound, however had a Kivalue of 10.4 nM. The increase in both adenosine A1 and A2A affinity can most likely be ascribed to the increase in nitrogens in the heterocyclic ring (from a dihydropyridine to a dihydropyrimidine) since similar results were obtained by Gillespie and co-workers in 2009 for a series of pyridine and pyrimidine derivatives. In their case it was found that increasing the number of nitrogens in the heterocyclic ring (from one to two nitrogen atoms for the pyridine and pyrimidine derivatives respectively) increased affinity for the adenosine A2A and adenosine A1 receptor subtypes, while three nitrogen atoms in the ring (triazine derivatives) were associated with decreased affinity. It thus appears that two nitrogen atoms in the ring (pyrimidine) are required for optimum adenosine A1 and A2A receptor affinity. The poor adenosine A2A affinity exhibited by the compounds of this study can probably be attributed to the absence of an aromatic heterocyclic ring. The amino acid, Phe-168 plays a very important role in the binding site of the A2A receptor, where it forms aromatic - - stacking interactions with the heterocyclic aromatic ring systems of known agonists and antagonists. Since the dihydropyrimidine ring in both series of this pilot study was not aromatic, the formation of aromatic - -stacking interactions with Phe-168 is unlikely. In conclusion, the 3,4-dihydropyrimidone and 2-amino-1,4-dihydropyrimidine scaffolds can be used as a lead for the design of novel adenosine A1 and A2A antagonists, although further structural modifications are required before a clinically viable candidate will be available as potential treatment of PD. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterised by a reduction of dopamine concentration in the striatum due to degeneration of dopaminergic neurons in the substantia nigra. Currently, first line treatment of PD includes the use of dopamine precursors, dopamine agonists and inhibitors of enzymatic degradation of dopamine, in an effort to restore dopamine levels and/or its effects. However, all these therapeutic strategies are only symptomatic and unfortunately do not slow, stop or reverse the progression of PD. From the discovery of adenosine A2A receptor-dopamine D2 receptor heteromers and the antagonistic interaction between these receptors, the basis of a new therapeutic approach towards the treatment of PD emerged. Adenosine A2A receptor antagonists have been shown to decrease the motor symptoms associated with PD, and are also potentially neuroprotective. The possibility thus exists that the administration of an adenosine A2A antagonist may prevent further neurodegeneration. Furthermore, the antagonism of adenosine A1 receptors has the potential of treating cognitive deficits such as those associated with Alzheimer's disease and PD. Therefore, dual antagonism of adenosine A1 and A2A receptors would be of great benefit since this would potentially treat both the motor as well as the cognitive impairment associated with PD. The affinities (Ki-values between 0.6 mM and 38 mM) of a series of 1,4-dihydropyridine derivatives were previously illustrated for the adenosine A1, A2A and A3 receptor subtypes by Van Rhee and co-workers (1996). These results prompted this pilot study, which aimed to investigate the potential of the structurally related 3,4-dihydropyrimidin-2(1H)-ones (dihydropyrimidones) and 2-amino-1,4-dihydropyrimidines as adenosine A1 and A2A antagonists. In this pilot study, a series of 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines were synthesised and evaluated as adenosine A1 and A2A antagonists. Since several adenosine A2A antagonists also exhibit MAO inhibitory activity, the MAO-inhibitory activity of selected derivatives was also assessed. A modified Biginelli one pot synthesis was used for the preparation of both series of compounds under solvent free conditions. A mixture of a β- diketone, aldehyde and urea/guanidine hydrochloride was heated for an appropriate time to afford the desired compounds in good yields. MAO-B inhibition studies comprised of a fluorometric assay where kynuramine was used as substrate. A radioligand binding protocol described in literature was employed to investigate the binding of the compounds to the adenosine A2A and A1 receptors. The displacement of N-[3H]ethyladenosin-5’-uronamide ([3H]NECA) from rat striatal membranes and 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat whole brain membranes, was used in the determination of A2A and A1 affinity, respectively. The results showed that both 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines had weak adenosine A2A affinity, with the p-fluorophenyl substituted dihydropyrimidone derivative (1h) in series 1, exhibiting the highest affinity for the adenosine A2A receptor (28.7 μM), followed by the p-chlorophenyl dihydropyrimidine derivative (2c) in series 2 (38.59 μM). Both series showed more promising adenosine A1 receptor affinity in the low micromolar range. The p-bromophenyl substituted derivatives in both series showed the best affinity for the adenosine A1 receptor with Ki-values of 7.39 μM (1b) and 7.9 μM (2b). The pmethoxyphenyl dihydropyrimidone (1d) and p-methylpneyl dihydropyrimidine (2e) derivatives also exhibited reasonable affinity for the adenosine A1 receptor with Ki-values of 8.53 μM and 9.67 μM, respectively. Neither the 3,4-dihydropyrimidones nor the 2-amino-1,4- dihydropyrimidines showed MAO-B inhibitory activity. Comparison of the adenosine A2A affinity of the most potent derivative (1h, Ki = 28.7 μM) from this study with that of the previously synthesised dihydropyridine derivatives (Van Rhee et al., 1996, most potent compound had a Ki = 2.74 mM) reveals that an approximate 100- fold increase in binding affinity for A2A receptors occurred. However, KW6002, a known A2A antagonist, that has already reached clinical trials, has a Ki-value of 7.49 nM. The same trend was observed for adenosine A1 affinity, where the most potent compound (1b) of this study exhibited a Ki-value of 7.39 μM compared to 2.75 mM determined for the most potent dihydropyridine derivatives (Van Rhee et al., 1996). N6-cyclopentyladenosine (CPA), a known adenosine A1 agonist that was used as a reference compound, however had a Kivalue of 10.4 nM. The increase in both adenosine A1 and A2A affinity can most likely be ascribed to the increase in nitrogens in the heterocyclic ring (from a dihydropyridine to a dihydropyrimidine) since similar results were obtained by Gillespie and co-workers in 2009 for a series of pyridine and pyrimidine derivatives. In their case it was found that increasing the number of nitrogens in the heterocyclic ring (from one to two nitrogen atoms for the pyridine and pyrimidine derivatives respectively) increased affinity for the adenosine A2A and adenosine A1 receptor subtypes, while three nitrogen atoms in the ring (triazine derivatives) were associated with decreased affinity. It thus appears that two nitrogen atoms in the ring (pyrimidine) are required for optimum adenosine A1 and A2A receptor affinity. The poor adenosine A2A affinity exhibited by the compounds of this study can probably be attributed to the absence of an aromatic heterocyclic ring. The amino acid, Phe-168 plays a very important role in the binding site of the A2A receptor, where it forms aromatic - - stacking interactions with the heterocyclic aromatic ring systems of known agonists and antagonists. Since the dihydropyrimidine ring in both series of this pilot study was not aromatic, the formation of aromatic - -stacking interactions with Phe-168 is unlikely. In conclusion, the 3,4-dihydropyrimidone and 2-amino-1,4-dihydropyrimidine scaffolds can be used as a lead for the design of novel adenosine A1 and A2A antagonists, although further structural modifications are required before a clinically viable candidate will be available as potential treatment of PD. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014