O adrenoceptor <font face=\"symbol\">b está presente na glândula de veneno de Bothrops jararaca, tanto no estado quiescente como no ativado por 4 dias, entretanto, quando estimulado, aumenta os níveis de AMPc somente em células secretoras no estado quiescente. Neste trabalho, estudamos funcionalmente o adrenoceptor <font face=\"symbol\">b durante o ciclo de produção de veneno e verificamos a sua função. Mostramos que a estimulação do adrenoceptor <font face=\"symbol\">b promove aumento na taxa de acidificação do meio extracelular em células quiescentes e ativadas por 4 dias, mas não por 15 dias. Em células quiescentes, a estimulação do adrenoceptor <font face=\"symbol\">b também promove o influxo de cálcio, via canais operados por voltagem e por receptor e a liberação de cálcio de estoques intracelulares sensíveis a tapsigargina. Em células ativadas por 4 dias, o aumento de cálcio citosólico é significativamente menor do que em células quiescentes, confirmando a dessensibilização deste receptor. PKA não participa desta sinalização. Dados in vivo mostram que o adrenoceptor <font face=\"symbol\">b é importante para desencadear o ciclo de produção de veneno. / <font face=\"symbol\">b adrenoceptor is present in quiescent and 4 days activated secretory cells of venom gland of Bothrops jararaca snake, however its stimulation increase cAMP production only in quiescent cells. In this work, we functionally characterized <font face=\"symbol\">b adrenoceptor during the venom production cycle and verified its function in venom production. We showed that the stimulation of <font face=\"symbol\">b adrenoceptor increases the rate of extracellular acidification in quiescent and 4 days activated cells, but not in 15 days activated cells. In quiescent cells, <font face=\"symbol\">b adrenoceptor stimulation also triggers calcium influx through voltage-operated and receptor-operated calcium channels, and calcium release from tapsigargin-sensitive stores. In 4 days activated cells, <font face=\"symbol\">b adrenoceptor stimulation promotes smaller increases in citosolic calcium concentration than in quiescent cells, confirming that this adrenoceptor undergoes to dessensitization. PKA does not participate in calcium signaling. In vivo experiments showed that <font face=\"symbol\">b adrenoceptor is important to trigger the cycle of venom production.

The aim of this thesis was to clarify the influence of the stress on the adrenergic and muscarinic receptors in the heart and in the lungs. Research was perform on rat hearts and lungs and on the hearts and lungs of the CRH KO mice. First, we assessed mRNA levels of all α- and β-adrenergic receptor and muscarinic receptor subtypes. Subsequently, we performed the radioligand-binding studies to determine densities of these receptors. We identified all three α1-adrenergic receptor subtypes in the rat lungs. In the lungs of WT mice, we found that the amount of α1-adrenergic and muscarinic receptors was sex-dependent. Densities of the former were higher in females and those of the latter were higher in males. There was no difference between males and females in β-adrenergic receptor density. As for CRH KO mice, the basal densities of studied receptors were lower than in CRH WT mice (except β1-adrenergic receptors in females). The main purpose of the thesis was to detect the immobilization-induced changes in the studied receptors in the kontrol (WT) and CRH KO mice. Short-term and long-term immobilization caused decrease in all α1-adrenergic receptor subtypes in females, whereas only α1A-adrenergic receptors decreased in males. The amount of β1-adrenergic receptors decreased in males and remained without...

Aims: To study the β-adrenoceptor subtypes involved in the relaxation responses to (-)-isoprenaline in carbachol-pre-contracted (CCh) mouse detrusor muscle with intact and denuded mucosa. Methods: Isolated muscle strips from the urinary bladder of male C57BL6 mice or β2-adrenoceptor knockout mice were pre-contracted with CCh, 1 µM and relaxed with increasing concentrations of the β-adrenoceptor (β-AR) agonist (-)-isoprenaline and forskolin. For estimating the β-AR subtypes involved, subtype-selective receptor blockers were used, that is, CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Unlike in KCl-pre-contracted muscle, the mucosa did not affect the sensitivity of the relaxation response to (-)-isoprenaline in CCh-pre-contracted murine detrusor strips. Increasing concentrations of (-)-isoprenaline produced a biphasic concentration-relaxation response without any difference both during the presence and absence of mucosa. The relaxation fraction produced by low (-)-isoprenaline concentrations was mediated by β2-AR as evidenced by a shift of the concentration-response curve to higher concentrations with ICI 118,551, but not with CGP 20712A and L748,337, and by the absence of this fraction in β2-AR-KO mice. The relaxation response with low sensitivity to (-)-isoprenaline was not affected by any of the β-AR subtype-selective blockers and was the only response detected in detrusor strips from β2-AR-KO mice. Conclusions: In CCh-pre-contracted mouse detrusor, β2-ARs are responsible for the relaxation component with high sensitivity to (-)-isoprenaline as indicated by the conversion of a biphasic into a monophasic CRC with ICI 118,551 or by its absence in β2-AR KO mice. The mucosa does not impair relaxation under these conditions. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

O estresse parece ser um dos principais fatores responsáveis pela predisposição do indivíduo à depressão. Estudos prévios de nosso laboratório mostraram que a inativação aguda da transmissão sináptica no núcleo leito da estria terminal (NLET) provoca efeitos tipo-antidepressivos em animais submetidos ao modelo animal do nado forçado, indicando que sua ativação durante o estresse facilita o desenvolvimento de consequências comportamentais relacionadas à neurobiologia da depressão. Contudo, os neurotransmissores envolvidos na mediação de tais efeitos permanecem desconhecidos. Sabe-se que a neurotransmissão noradrenérgica no NLET é abundante e tem participação importante na regulação de processos emocionais relacionados à resposta ao estresse. Assim, o presente trabalho investigou o envolvimento da neurotransmissão noradrenérgica existente no núcleo leito da estria terminal (NLET) no desenvolvimento das consequências comportamentais do estresse relacionadas à neurobiologia da depressão, através da administração local de antagonistas noradrenérgicos em animais submetidos ao modelo animal do nado forçado. Os resultados mostraram que a administração local de WB4101 (antagonista ?1) (10 e 15 nmol), CGP20712 (antagonista ?1) (5 e 10 nmol) e ICI118,551 (antagonista ?2) (5 nmol) reduziu o tempo de imobilidade de animais submetidos ao teste do nado forçado. Além disso, a administração local das mesmas doses dessas drogas em grupos independentes de animais submetidos ao teste do campo aberto não causou alteração na atividade locomotora dos animais, descartando um possível efeito inespecífico sobre a atividade locomotora. Os resultados sugerem, portanto, o envolvimento da neurotransmissão noradrenérgica no NLET, através da ativação de receptores noradrenérgicos ?1, ?1 e ?2, na mediação das consequências comportamentais do estresse e nos mecanismos neurais envolvidos na neurobiologia da depressão. / Stress seems to be a major factor responsible for the individual\'s predisposition to depression. Previous studies from our laboratory have shown that acute inactivation of synaptic transmission within the bed nucleus of the stria terminalis (NLET) with cobalt chloride causes antidepressant-like effects in rats submitted to the forced swimming test, suggesting that NLET activation during stress facilitates the development of the behavioral consequences related to the neurobiology of depression. However, the neurotransmitters involved in mediating these effects remain unknown. It is known that the noradrenergic neurotransmission in NLET is abundant and plays an important role in regulating emotional processes related to stress response. Thus, the present study investigated the involvement of the noradrenergic neurotransmission in the bed nucleus of the stria terminalis (NLET) in the development of the behavioral consequences of stress related to the neurobiology of depression by local administration of noradrenergic antagonists in rats submitted to the forced swimming animal model. The results showed that the local administration of WB4101 (?1-antagonist) (10 and 15 nmol), CGP20712 (?1-antagonist) (5 and 10 nmol) and ICI118, 551 (?2-antagonist) (5 nmol) reduced the immobility time of animals submitted to the forced swimming test, an antidepressant-like effect. Furthermore, local administration of the same doses of these drugs in independent groups of animals submitted to the open field test did not change their locomotor activity, discarding a possible unspecific effect on locomotor activity. The results, therefore, suggest the involvement of the noradrenergic neurotransmission within the NLET, through the activation of noradrenergic receptors ?1, ?1 and ?2, in mediating the behavioral consequences of stress and the neural mechanisms involved in the neurobiology of depression.

O estresse parece ser um dos principais fatores responsáveis pela predisposição do indivíduo à depressão. Estudos prévios de nosso laboratório mostraram que a inativação aguda da transmissão sináptica no núcleo leito da estria terminal (NLET) provoca efeitos tipo-antidepressivos em animais submetidos ao modelo animal do nado forçado, indicando que sua ativação durante o estresse facilita o desenvolvimento de consequências comportamentais relacionadas à neurobiologia da depressão. Contudo, os neurotransmissores envolvidos na mediação de tais efeitos permanecem desconhecidos. Sabe-se que a neurotransmissão noradrenérgica no NLET é abundante e tem participação importante na regulação de processos emocionais relacionados à resposta ao estresse. Assim, o presente trabalho investigou o envolvimento da neurotransmissão noradrenérgica existente no núcleo leito da estria terminal (NLET) no desenvolvimento das consequências comportamentais do estresse relacionadas à neurobiologia da depressão, através da administração local de antagonistas noradrenérgicos em animais submetidos ao modelo animal do nado forçado. Os resultados mostraram que a administração local de WB4101 (antagonista ?1) (10 e 15 nmol), CGP20712 (antagonista ?1) (5 e 10 nmol) e ICI118,551 (antagonista ?2) (5 nmol) reduziu o tempo de imobilidade de animais submetidos ao teste do nado forçado. Além disso, a administração local das mesmas doses dessas drogas em grupos independentes de animais submetidos ao teste do campo aberto não causou alteração na atividade locomotora dos animais, descartando um possível efeito inespecífico sobre a atividade locomotora. Os resultados sugerem, portanto, o envolvimento da neurotransmissão noradrenérgica no NLET, através da ativação de receptores noradrenérgicos ?1, ?1 e ?2, na mediação das consequências comportamentais do estresse e nos mecanismos neurais envolvidos na neurobiologia da depressão. / Stress seems to be a major factor responsible for the individual\'s predisposition to depression. Previous studies from our laboratory have shown that acute inactivation of synaptic transmission within the bed nucleus of the stria terminalis (NLET) with cobalt chloride causes antidepressant-like effects in rats submitted to the forced swimming test, suggesting that NLET activation during stress facilitates the development of the behavioral consequences related to the neurobiology of depression. However, the neurotransmitters involved in mediating these effects remain unknown. It is known that the noradrenergic neurotransmission in NLET is abundant and plays an important role in regulating emotional processes related to stress response. Thus, the present study investigated the involvement of the noradrenergic neurotransmission in the bed nucleus of the stria terminalis (NLET) in the development of the behavioral consequences of stress related to the neurobiology of depression by local administration of noradrenergic antagonists in rats submitted to the forced swimming animal model. The results showed that the local administration of WB4101 (?1-antagonist) (10 and 15 nmol), CGP20712 (?1-antagonist) (5 and 10 nmol) and ICI118, 551 (?2-antagonist) (5 nmol) reduced the immobility time of animals submitted to the forced swimming test, an antidepressant-like effect. Furthermore, local administration of the same doses of these drugs in independent groups of animals submitted to the open field test did not change their locomotor activity, discarding a possible unspecific effect on locomotor activity. The results, therefore, suggest the involvement of the noradrenergic neurotransmission within the NLET, through the activation of noradrenergic receptors ?1, ?1 and ?2, in mediating the behavioral consequences of stress and the neural mechanisms involved in the neurobiology of depression.

Made available in DSpace on 2016-06-02T19:22:12Z (GMT). No. of bitstreams: 1 6524.pdf: 836310 bytes, checksum: 7abc8c7790ee59b230df28eb660ac2b7 (MD5) Previous issue date: 2009-12-17 / Universidade Federal de Sao Carlos / Lipopolysaccharide (LPS), an endotoxin derived from gram-negative bacteria wall, triggers a conjunct of systemic and behavioral responses grouped under the name of sickness behavior. Several works show the inhibition of ingestive behavior in animals with activated immune system by systemic LPS. The aim of this work was demonstrate that LPS inhibit sodium appetite in lower doses than those that cause lethal effects and that LPS inhibitory action on sodium appetite involves &#945;2 adrenergic mechanisms. Were produced three works where we show the LPS effects on sodium appetite, on arterial pressure and in different kinds of thirst. In the first work, we showed that LPS inhibited sodium appetite and only higher doses produced thirst inhibition, besides reduced urinary volume and natriuresis, without alterations on cardiovascular parameters in FURO/CAP-treated rats. LPS abolished intracellular thirst e reduced urinary volume and natriuresis in 2M NaCl gavage-treated animals. LPS also reduced thirst in FURO-treated animals. These results suggest that LPS has a preferential effect on sodium appetite and on thirst intracellular thirst. In the second work we showed that LPS reduce sodium appetite and that this inhibition is abolished by previous treatment with yohimbine ip, an &#945;2 adrenoceptor antagonist in sodium depleted rats. Yohimbine when combined with LPS produced hypotension in sodium depleted rats. LPS reduced gastric empty in sodium depleted rats and this effect wasn&#8223;t abolished by previous treatment with yohimbine. The systemic effects do not explain the inhibition on sodium appetite and we can suggest that LPS exert an inhibitory effect on sodium appetite probably mediated by central &#945;2 adrenoceptors. In the third work we confirmed that LPS inhibit sodium appetite and that central &#945;2 adrenergic ways mediated this response because RX-821002, an &#945;2 adrenoceptor antagonist injected icv, abolished sodium appetite inhibition induced by systemic LPS in sodium depleted rats without produced alterations in cardiovascular parameters. All results exposed with these three works suggest that LPS inhibit sodium appetite and this inhibition is mediated by central &#945;2 adrenoceptors. / Lipopolissacarídeo (LPS), uma endotoxina derivada da parede da bactéria gram-negativa, desencadeia um conjunto de respostas sistêmicas e comportamentais, que se agrupam sob a denominação de comportamento de doença (sickness behavior). Grande número de trabalhos mostra a inibição de comportamento ingestivo no animal que tem ativação do sistema imune induzido por injeção sistêmica de LPS. O objetivo deste trabalho foi demonstrar que o LPS inibe o apetite ao sódio em doses menores daquelas que causam os efeitos letais desta endotoxina e que a ação inibitória do LPS sobre o apetite ao sódio envolve mecanismos adrenérgicos &#945;2. Foram produzidos três trabalhos onde mostramos os efeitos do LPS no apetite ao sódio, na pressão arterial e em diferentes tipos de sede. No primeiro trabalho mostramos que LPS inibiu o apetite ao sódio e apenas as doses mais altas produziram inibição na sede, além de reduzir volume urinário e natriurese, sem alterar os parâmetros cardiovasculares em ratos tratados com o protocolo FURO/CAP. LPS aboliu sede intracelular e reduziu volume urinário e natriurese em ratos que receberam gavagem de NaCl 2M. A sede também foi reduzida pelo tratamento com LPS em ratos tratados com FURO apenas. Esses resultados sugerem que o LPS tem efeito preferencial sobre o apetite ao sódio e sobre a sede intracelular. No segundo trabalho mostramos que LPS reduz o apetite ao sódio e que essa inibição é abolida se há tratamento prévio com ioimbina ip, um antagonista de receptores adrenérgicos &#945;2 em ratos depletados de sódio. Esse mesmo antagonista quando associado com LPS produz hipotensão em ratos depletados de sódio. LPS diminuiu a taxa de esvaziamento gástrico em animais depletados de sódio e esse efeito não foi abolido pelo prévio tratamento com ioimbina. Devido ao fato desses efeitos sistêmicos do LPS não explicarem a inibição no apetite ao sódio podemos sugerir que o LPS exerce um efeito inibitório no apetite ao sódio provavelmente mediado por receptores adrenérgicos &#945;2 centrais. No terceiro trabalho confirmamos que o LPS inibe o apetite ao sódio e que vias centrais noradrenérgicas &#945;2 centrais medeiam essa inibição pois o RX-821002, um antagonista de receptores adrenérgicos &#945;2, injetado icv, também aboliu a inibição no apetite ao sódio induzida pelo LPS sistêmico em ratos depletados de sódio sem produzir alterações nos parâmetros cardiovasculares. Esse conjunto de resultados expostos por meio destes três trabalhos sugere que o LPS inibe o apetite ao sódio e essa inibição é mediada por receptores adrenérgicos &#945;2 de origem central.

Während der letzten Jahre zeigten einige Studien, dass die Behandlung mit Knochenmark- Stammzellen (KMSZ) eine vielversprechende neue Therapieoption für den geschädigten Herzmuskel darstellen könnte. In dieser Arbeit wurde untersucht, ob es unter Behandlung mit Stammzellen zu einer zellulären Antwort in angezüchteten Kardiomyozyten (KMZ) kommt. Dafür wurden subkonfluente Kulturen aus Herzmuskelzellen von neonatalen Ratten für drei Tage mit Vybrant CM-DiI-markierten, sternalen humanen Knochenmarkstammzellen co-kultiviert. Im Anschluss wurden immunohistochemische Färbungen sowie eine quantitative Analyse mittels Western Blot für das Protoonkogen c-Myc durchgeführt. Des Weiteren wurde die Dichte der Beta-Adrenozeptoren unter Anwendung einer Histoautoradiographie mittels [125I]- iodocyanopindolol-Bindung analysiert. Die Auswertung der Immunohistochemie und der Western Blots zeigte eine signifikante Erhöhung der Expression von c-Myc in den Kardiomyozyten, welche in naher Umgebung der Stammzellen lagen. Dieser Effekt war direkt abhängig von der Entfernung der KMZ zur SZ. Die Histoautoradiographie zeigte eine signifikant höhere Beta-Rezeptor-Dichte in Kardiomyozyten in direkter Nähe zur Stammzelle. Mit steigender Entfernung von der Stammzelle verringerte sich die Rezeptordichte. Somit konnte gezeigt werden, dass eine kleine Anzahl von Knochenmark-Stammzellen ausreicht, um eine große Zahl von Kardiomyozyten zu beeinflussen, indem eine intrazelluläre Signalkaskade über c-Myc aktiviert und die Anzahl der Beta-Adrenozeptoren erhöht wird.

Aims: To study the β-adrenoceptor subtypes involved in the relaxation responses to (-)-isoprenaline in carbachol-pre-contracted (CCh) mouse detrusor muscle with intact and denuded mucosa. Methods: Isolated muscle strips from the urinary bladder of male C57BL6 mice or β2-adrenoceptor knockout mice were pre-contracted with CCh, 1 µM and relaxed with increasing concentrations of the β-adrenoceptor (β-AR) agonist (-)-isoprenaline and forskolin. For estimating the β-AR subtypes involved, subtype-selective receptor blockers were used, that is, CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Unlike in KCl-pre-contracted muscle, the mucosa did not affect the sensitivity of the relaxation response to (-)-isoprenaline in CCh-pre-contracted murine detrusor strips. Increasing concentrations of (-)-isoprenaline produced a biphasic concentration-relaxation response without any difference both during the presence and absence of mucosa. The relaxation fraction produced by low (-)-isoprenaline concentrations was mediated by β2-AR as evidenced by a shift of the concentration-response curve to higher concentrations with ICI 118,551, but not with CGP 20712A and L748,337, and by the absence of this fraction in β2-AR-KO mice. The relaxation response with low sensitivity to (-)-isoprenaline was not affected by any of the β-AR subtype-selective blockers and was the only response detected in detrusor strips from β2-AR-KO mice. Conclusions: In CCh-pre-contracted mouse detrusor, β2-ARs are responsible for the relaxation component with high sensitivity to (-)-isoprenaline as indicated by the conversion of a biphasic into a monophasic CRC with ICI 118,551 or by its absence in β2-AR KO mice. The mucosa does not impair relaxation under these conditions. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Purpose: 5-hydroxytryptamine (5-HT) is an important neurotransmitter in depression. It is believed that α 1 and α 2 adrenoceptors mediate the 5-HT level in the brain. The mechanism is complex and not well explored. Especially in different combination treatments, the receptor systems may show varied modulation capability. Additionally, some research has suggested that α 2 heteroceptors may contribute to the time delay problem in dual depression treatment which is thought as the time needed for certain inhibition receptor to get desensitized. We hypothesized that the α 2 adrenoceptors had inhibition effect on 5-HT level in dorsal raphé nucleus (DRN), Prefrontal cortex (PFC) and Hippocampus (HP) with the dual reuptake inhibition. The present study was undertaken to explore the effect of BRL44408 (α 2 receptor antagonist) on 5-HT level in rat PFC, DRN and HP under dual antidepressant with blocking the α 1 receptor. Method: Serotonin reuptake inhibitor and norepinephrine reuptake inhibitor were used to mimic the dual reuptake inhibition antidepressant. To differentiate the α 2 adrenoceptors effect from al adrenoceptors effect, prazosin, an antagonist of α 1 adrenoceptors, was added to block α 1 adrenoceptors. Using the microdialysis method, the drug combination was examined in HP area and then DRN area to explore the drug effect on time course of 5-HT release in DRN and PFC. Based on the experiment results from DRN and PFC, a mechanism-based pharmacodynamic model was developed. Result: BRL44408 increased the serotonin (5-HT) level in rat PFC, DRN and HP to different degrees with the dual reuptake inhibition (p < 0.05). The overall model reasonably captured the time course of 5-HT in both DRN and PFC with different dose schemes of BRL44408. The model predicted EC50 of BRL44408 (0.0075 µM) for the α 2 heteroreceptor which control PFC 5-HT is close to the reported value of BRL44408 for α 2 adrenorceptor (0.008 µM). However, the model predicted EC50 of BRL44408 on the α 2 heteroreceptor which control DRN 5-HT need to be explained. Simulation result from this model suggested varied modulation capability of α 2 adrenoceptors on the 5-HT in DRN and the 5-HT in PFC. Conclusion: α 2 heteroceptor play a role in regulation 5-HT level under dual reuptake inhibition. Further exploration may bring a potential target for depression treatment. The mechanism model was developed to characterize and better understand the neurotransmitter mechanisms, providing estimations of various parameters of the disease related receptor system.

Objectives: The mucosa of human detrusor strips impairs catecholamine-induced relaxation. In order to elucidate which signal transduction pathways are involved in this cross talk between the mucosa and detrusor, we have studied the effects of several pharmacological agonists and antagonists on noradrenaline-mediated relaxation in intact and mucosa-denuded detrusor strips. Patients and Methods: Strips of detrusor tissue were obtained from patients who had undergone cystectomy for bladder cancer and were set up for force measurement. KCl- or carbachol-precontracted strips were relaxed with increasing concentrations of noradrenaline in the absence and in the presence of nitric oxide synthase inhibitor, L-NAME; P2X-receptor antagonist, PPADS; ET A -receptor antagonist, BQ-123; ET B -receptor antagonist, BQ-788; cyclooxygenase inhibitor, diclofenac; AT 1 -receptor antagonist, candesartan; and NK 1 -receptor antagonist, L-703,606. Results: In intact strips, KCl-stimulated force was enhanced by all blockers; carbachol-stimulated force increased with L-703,606. In denuded strips, only L-NAME augmented the KCl-stimulated contraction. Noradrenaline relaxed the precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. L-NAME, PPADS and BQ-123/BQ-788 had little effect on noradrenaline-induced relaxation, whereas diclofenac, candesartan and L-703,606 sensitized intact carbachol-stimulated detrusor strips to noradrenaline-induced relaxation. Conclusion: Inhibition of the noradrenaline-induced relaxation of precontracted human detrusor strips by the mucosa is attenuated by diclofenac, candesartan and L-703,606 suggesting the involvement of prostanoids, angiotensin and neurokinin pathways. Further experiments are required to unravel the exact mechanisms.