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Orientador: Patricia Maria de Paula / Banca: Carina Aparecida Fabrício de Andrade / Banca: Juliana Irani Fratucci de Gobbi / Resumo: Vários estudos demonstraram que o controle da ingestão hidro-mineral envolve diversos mediadores no sistema nervoso central. Entretanto a participação da neurotransmissão purinérgica no controle da ingestão de água e sódio ainda é pouco conhecida. Estudos imunohistoquímicos evidenciam a densa marcação de receptores purinérgicos em várias regiões envolvidas com o controle hidro-mineral, dentre elas o núcleo do trato solitário (NTS). O NTS possui mecanismos inibitórios envolvidos no controle da ingestão de sódio e água. Desta forma, os objetivos deste estudo foram estudar a participação dos receptores purinérgicos do NTS: 1) no controle da ingestão de água e sódio hipertônico (apetite ao sódio) em ratos com depleção de sódio (produzida pelo tratamento com diurético furosemida); 2) no controle da ingestão de água em ratos com privação hídrica de 24 horas (modelo de desidratação extra- e intracelular) e 3) no controle da ingestão de água em ratos com sobrecarga intragástrica de NaCl 12% (modelo de desidratação intracelular). Foram utilizados ratos Holtzman (280-320 g) com cânulas guias bilaterais implantadas no NTS. Injeções bilaterais de alfa-beta-metileno-ATP (α,β-metil ATP, agonista purinérgico P2X, 1,0 nmol/0,1 μl) ou suramin (antagonista purinérgico não seletivo P2, 1,0 nmol/0,1 μl) no NTS não alteraram a ingestão de NaCl 1.8% induzida pela depleção de sódio (15,4 ± 1,7 ml/2 h vs salina 19,7 ± 1,7 ml/2 h e 18,9 ± 2,7 vs. salina 22,7 ± 2,3 ml/2 h, respectivamente,). A ingestão de água induzida pela privação hídrica também não foi alterada pelo agonista purinérgico α,β-metil ATP [1,0 ou 2,0 nmol/0,1 μl (15,0 ± 0,9 vs. salina 14,8 ± 1,0 ml/2 h e 16,8 ± 0,7 vs. salina 17,7 ± 0,9 ml/2 h, respectivamente)] ou pelos antagonistas purinérgicos suramin [1,0 nmol/0,1 μl (15,1 ± 0,9 vs. salina 14,8 ± 1,0 ml/2 h)]... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Several studies have shown that the hydro-mineral intake control involves different mediators in the central nervous systems. However, the involvement of purinergic neurotransmission in the control of water and sodium intake is still unknown. Immunohistochemical studies showed a dense labeling of purinergic receptors in many regions involved in the control of the hydro-mineral balance, among them the nucleus of the solitary tract (NTS). The NTS has inhibitory mechanisms involved in the control of sodium and water intake. Thus, the aims of this study were to investigate the involvement of purinergic receptors of the NTS: 1) in the control of water and hypertonic sodium (sodium appetite) intake in rats with sodium depletion (produced by treatment with diuretic furosemide), 2) in the control of water intake in rats subtracted to 24h water deprivation (a model that mixes extra- and intracellular dehydration) and 3) in the control of water intake in rats treated with intragastric gavage with 2 ml of 12% NaCl (a model of intracellular dehydration). Male Holtzman rats (280-320 g) with bilateral guide cannulas implanted into the NTS were used. Bilateral injections of alpha-beta-methylene ATP (α,β-methyl ATP, P2X purinergic agonist, 1.0 nmol/0.1 μl) or suramin (non-selective P2 purinergic antagonist, 1.0 nmol/0.1 μl) into the NTS did not change the 1.8% NaCl intake induced by sodium depletion (15.4 ± 1.7 ml/2h vs saline 19.7 ± 1.7 ml/2h and 18.9 ± 2.7 vs. saline 22.7 ± 2.3 ml/2h, respectively). Water intake induced by water deprivation was not altered by purinergic agonist α,β-methyl ATP [1.0 or 2.0 nmol/0.1 μl (15.0 ± 0.9 vs. saline 14.8 ± 1.0 ml/2 h and 16.8 ± 0.7 vs. saline 17.7 ± 0.9 ml/2 h, respectively)] or by the purinergic antagonists suramin [1.0 nmol/0.1 μl (15.1 ± 0.9 vs. saline 14.8 ± 1.0 ml/2 h)] or PPADS [ 1, 2, 3 and 4.0 nmol/0.1 ul, purinergic P2X antagonist... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:30:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-06Bitstream added on 2014-06-13T20:00:26Z : No. of bitstreams: 1 tomeo_ra_me_arafo.pdf: 932626 bytes, checksum: 3355361fef26b03348e905ee0beb1a79 (MD5) / Vários estudos demonstraram que o controle da ingestão hidro-mineral envolve diversos mediadores no sistema nervoso central. Entretanto a participação da neurotransmissão purinérgica no controle da ingestão de água e sódio ainda é pouco conhecida. Estudos imunohistoquímicos evidenciam a densa marcação de receptores purinérgicos em várias regiões envolvidas com o controle hidro-mineral, dentre elas o núcleo do trato solitário (NTS). O NTS possui mecanismos inibitórios envolvidos no controle da ingestão de sódio e água. Desta forma, os objetivos deste estudo foram estudar a participação dos receptores purinérgicos do NTS: 1) no controle da ingestão de água e sódio hipertônico (apetite ao sódio) em ratos com depleção de sódio (produzida pelo tratamento com diurético furosemida); 2) no controle da ingestão de água em ratos com privação hídrica de 24 horas (modelo de desidratação extra- e intracelular) e 3) no controle da ingestão de água em ratos com sobrecarga intragástrica de NaCl 12% (modelo de desidratação intracelular). Foram utilizados ratos Holtzman (280-320 g) com cânulas guias bilaterais implantadas no NTS. Injeções bilaterais de alfa-beta-metileno-ATP (α,β-metil ATP, agonista purinérgico P2X, 1,0 nmol/0,1 μl) ou suramin (antagonista purinérgico não seletivo P2, 1,0 nmol/0,1 μl) no NTS não alteraram a ingestão de NaCl 1.8% induzida pela depleção de sódio (15,4 ± 1,7 ml/2 h vs salina 19,7 ± 1,7 ml/2 h e 18,9 ± 2,7 vs. salina 22,7 ± 2,3 ml/2 h, respectivamente,). A ingestão de água induzida pela privação hídrica também não foi alterada pelo agonista purinérgico α,β-metil ATP [1,0 ou 2,0 nmol/0,1 μl (15,0 ± 0,9 vs. salina 14,8 ± 1,0 ml/2 h e 16,8 ± 0,7 vs. salina 17,7 ± 0,9 ml/2 h, respectivamente)] ou pelos antagonistas purinérgicos suramin [1,0 nmol/0,1 μl (15,1 ± 0,9 vs. salina 14,8 ± 1,0 ml/2 h)]... / Several studies have shown that the hydro-mineral intake control involves different mediators in the central nervous systems. However, the involvement of purinergic neurotransmission in the control of water and sodium intake is still unknown. Immunohistochemical studies showed a dense labeling of purinergic receptors in many regions involved in the control of the hydro-mineral balance, among them the nucleus of the solitary tract (NTS). The NTS has inhibitory mechanisms involved in the control of sodium and water intake. Thus, the aims of this study were to investigate the involvement of purinergic receptors of the NTS: 1) in the control of water and hypertonic sodium (sodium appetite) intake in rats with sodium depletion (produced by treatment with diuretic furosemide), 2) in the control of water intake in rats subtracted to 24h water deprivation (a model that mixes extra- and intracellular dehydration) and 3) in the control of water intake in rats treated with intragastric gavage with 2 ml of 12% NaCl (a model of intracellular dehydration). Male Holtzman rats (280-320 g) with bilateral guide cannulas implanted into the NTS were used. Bilateral injections of alpha-beta-methylene ATP (α,β-methyl ATP, P2X purinergic agonist, 1.0 nmol/0.1 μl) or suramin (non-selective P2 purinergic antagonist, 1.0 nmol/0.1 μl) into the NTS did not change the 1.8% NaCl intake induced by sodium depletion (15.4 ± 1.7 ml/2h vs saline 19.7 ± 1.7 ml/2h and 18.9 ± 2.7 vs. saline 22.7 ± 2.3 ml/2h, respectively). Water intake induced by water deprivation was not altered by purinergic agonist α,β-methyl ATP [1.0 or 2.0 nmol/0.1 μl (15.0 ± 0.9 vs. saline 14.8 ± 1.0 ml/2 h and 16.8 ± 0.7 vs. saline 17.7 ± 0.9 ml/2 h, respectively)] or by the purinergic antagonists suramin [1.0 nmol/0.1 μl (15.1 ± 0.9 vs. saline 14.8 ± 1.0 ml/2 h)] or PPADS [ 1, 2, 3 and 4.0 nmol/0.1 ul, purinergic P2X antagonist... (Complete abstract click electronic access below)

The effects of limited and infrequent drinking, and atropine administration on feed intake and utilization was investigated in South African indigenous goats. Sixteen goats with an average body weight of 29.1 kg were subjected to water restriction and deprivation with concurrent atropine administration. They were fed ad libitum with a mixture of lucerne (Medicago sativa) and eragrostis hay (Eragrostis curvula), blended with molasses. The diet contained 10.47% crude protein, 38% crude fibre and 17.5 MJ/kg gross energy. Fifteen goats were randomly divided into 3 groups and were watered ad Libitum, 50% of ad libitum and 30% of ad libitum water intake respectively (Trial 1). In trial 2, a group of 8 animals were deprived of water for 3 days while the other group had free access to water daily (phase1). During phase 2, another group of 8 were watered on the 5th day while others had water ad libitum. A subgroup of 4 goats each were injected with atropine in both phases. The results showed that these goats have high water efficiency. The limited and infrequent supply of water decreased feed intake but enhanced nutrient utilisation. The provision of water at the 50% ad libitum level or once in 3 days is economical and beneficial to goat production in water-scarce areas. There is a need for complimentary investigations using atropine at high doses to further elucidate the effects of this drug on the gastro-intestinal functions of ruminants. / Dissertation (MSc (Physiology))--University of Pretoria, 2007. / Anatomy and Physiology / unrestricted

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.

O Chronic Mild Stress (CMS) é um modelo animal de depressão estudado para investigação das causas e dos atenuadores deste problema. Nele, ratos são submetidos a um protocolo de estressores moderadamente aversivos de maneira crônica. É observada diminuição na ingestão e preferência por substância líquida doce por conta desta submissão. Em estudo sobre o papel da privação de alimento e água no conjunto de estressores, Pereira e Sério (2010) não observaram os efeitos de diminuição na ingestão e preferência por sacarose. É levantada a possibilidade de que a privação de água e alimento a que os sujeitos foram submetidos previamente ao protocolo poderia ter sido responsável pelas diferenças encontradas, já que diferiu em muitos aspectos do realizado por Willner et al. (1987). O objetivo do presente estudo foi o de investigar o papel da privação de água no protocolo de estressores. Três grupos de sujeitos foram propostos: Grupo Protocolo (submetido ao protocolo completo), Grupo Privação (submetido apenas à privação de água presente no protocolo) e Grupo Controle (submetido apenas aos testes semanais de ingestão e preferência). Foram realizados testes de ingestão e preferência por sacarose antes, durante e depois do protocolo ou da privação e aferições semanais de peso e consumo de alimento e água. Como resultado o consumo de sacarose apresentou queda com o decorrer dos testes tanto para os Grupos Protocolo e Privação quanto para o Grupo Controle. Ou seja, não é possível atribuir a nenhum dos procedimentos (protocolo ou privação) a queda observada nessa medida. O peso corporal apresentou queda também para os três grupos assim que iniciado o procedimento de privação precedente ao teste. Apenas quando iniciados o protocolo e a privação para os grupos correspondentes, ocorreu menor ganho de peso semanal nesses grupos em comparação ao Grupo Controle. Com relação ao consumo de alimento, observou-se queda para os três grupos quando iniciada a privação semanal. Iniciado o protocolo de estressores, o consumo de alimento para o Grupo Protocolo apresentou a menor queda dentre os três, comparativamente ao que consumia antes. É sugerido que o consumo de sacarose não é uma boa medida dos efeitos do protocolo, sendo discutidas possibilidades de maior controle sobre essa medida e sugeridas outras medidas como possivelmente mais precisas para aferir os efeitos do protocolo / Chronic Mild Stress (CMS) is an animal model of depression studied by different areas of research into the causes and \"attenuators\" of this problem. In it, rats are subjected to a protocol of moderately aversive stressors applied chronicaly. Decreased intake and preference for sweet liquid substance on behalf of that submission is observed. In a study on the role of the deprivation of food and water in the set of stressors, Pereira and Sério (2010) did not observe the effects of decreased intake and preference for sucrose. It is raised the possibility that the deprivation of food and water that the subjects were submitted previously to the protocol might have been responsible for the differences found, given it differed in many aspects to what was done by Willner et al. (1987). The aim of the present study was to investigate the role of water deprivation in the stress protocol. Three groups of subjects were proposed: Protocol (submitted to the full protocol), Deprivation (subject only to the deprivation of water present in the protocol) and Control (subject only to the weekly consumption and preference tests). Free tests of sucrose intake and preference before, during and after the protocol or the deprivation were carried out. The main results observed were that for both the Protocol and Deprivation Groups and for the Control Group, sucrose consumption decreased over the course of the tests. That means it is not possible to assign to any of the procedures (protocol or deprivation) the decreased consumption observed. Body weight decreased in the three groups as soon as the deprivation procedure preceding the test started, but when the protocol and deprivation for the corresponding groups began, lower weekly weight gain occurred in these groups compared to the control group. With respect to food intake, a decrease for the three groups when initiated weekly deprivation was observed. As the stress protocol began, food intake in this group had the smallest drop compared to that consumed before in relation to the other groups. It is suggested therefore that the sucrose consumption is not a good measure of the effects of the protocol, and possibilities of control over this measure are discussed, along with the suggestion of other more precise measures of the effects of the stress protocol

O Chronic Mild Stress (CMS) é um modelo animal de depressão estudado para investigação das causas e dos atenuadores deste problema. Nele, ratos são submetidos a um protocolo de estressores moderadamente aversivos de maneira crônica. É observada diminuição na ingestão e preferência por substância líquida doce por conta desta submissão. Em estudo sobre o papel da privação de alimento e água no conjunto de estressores, Pereira e Sério (2010) não observaram os efeitos de diminuição na ingestão e preferência por sacarose. É levantada a possibilidade de que a privação de água e alimento a que os sujeitos foram submetidos previamente ao protocolo poderia ter sido responsável pelas diferenças encontradas, já que diferiu em muitos aspectos do realizado por Willner et al. (1987). O objetivo do presente estudo foi o de investigar o papel da privação de água no protocolo de estressores. Três grupos de sujeitos foram propostos: Grupo Protocolo (submetido ao protocolo completo), Grupo Privação (submetido apenas à privação de água presente no protocolo) e Grupo Controle (submetido apenas aos testes semanais de ingestão e preferência). Foram realizados testes de ingestão e preferência por sacarose antes, durante e depois do protocolo ou da privação e aferições semanais de peso e consumo de alimento e água. Como resultado o consumo de sacarose apresentou queda com o decorrer dos testes tanto para os Grupos Protocolo e Privação quanto para o Grupo Controle. Ou seja, não é possível atribuir a nenhum dos procedimentos (protocolo ou privação) a queda observada nessa medida. O peso corporal apresentou queda também para os três grupos assim que iniciado o procedimento de privação precedente ao teste. Apenas quando iniciados o protocolo e a privação para os grupos correspondentes, ocorreu menor ganho de peso semanal nesses grupos em comparação ao Grupo Controle. Com relação ao consumo de alimento, observou-se queda para os três grupos quando iniciada a privação semanal. Iniciado o protocolo de estressores, o consumo de alimento para o Grupo Protocolo apresentou a menor queda dentre os três, comparativamente ao que consumia antes. É sugerido que o consumo de sacarose não é uma boa medida dos efeitos do protocolo, sendo discutidas possibilidades de maior controle sobre essa medida e sugeridas outras medidas como possivelmente mais precisas para aferir os efeitos do protocolo / Chronic Mild Stress (CMS) is an animal model of depression studied by different areas of research into the causes and \"attenuators\" of this problem. In it, rats are subjected to a protocol of moderately aversive stressors applied chronicaly. Decreased intake and preference for sweet liquid substance on behalf of that submission is observed. In a study on the role of the deprivation of food and water in the set of stressors, Pereira and Sério (2010) did not observe the effects of decreased intake and preference for sucrose. It is raised the possibility that the deprivation of food and water that the subjects were submitted previously to the protocol might have been responsible for the differences found, given it differed in many aspects to what was done by Willner et al. (1987). The aim of the present study was to investigate the role of water deprivation in the stress protocol. Three groups of subjects were proposed: Protocol (submitted to the full protocol), Deprivation (subject only to the deprivation of water present in the protocol) and Control (subject only to the weekly consumption and preference tests). Free tests of sucrose intake and preference before, during and after the protocol or the deprivation were carried out. The main results observed were that for both the Protocol and Deprivation Groups and for the Control Group, sucrose consumption decreased over the course of the tests. That means it is not possible to assign to any of the procedures (protocol or deprivation) the decreased consumption observed. Body weight decreased in the three groups as soon as the deprivation procedure preceding the test started, but when the protocol and deprivation for the corresponding groups began, lower weekly weight gain occurred in these groups compared to the control group. With respect to food intake, a decrease for the three groups when initiated weekly deprivation was observed. As the stress protocol began, food intake in this group had the smallest drop compared to that consumed before in relation to the other groups. It is suggested therefore that the sucrose consumption is not a good measure of the effects of the protocol, and possibilities of control over this measure are discussed, along with the suggestion of other more precise measures of the effects of the stress protocol