The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis. / Ph. D.

Yes / Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.

Tyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic and spectroscopic techniques was applied in order to obtain insight into the catalytic mechanism of this physiologically important enzyme. Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies established the kinetic competency of this intermediate to be the long-postulated hydroxylating species, Fe(IV)O. Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS) showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However, when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that there is a significant enhancement in reactivity in the 5-coordinate complex in comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed that Glu332 residue plays a role in directing the protonation of the bridged complex that forms prior to the formation of Fe(IV)O. Rapid chemical quench analyses of DOPA formation showed a burst of product formation, suggesting a slow product release step. Steady-state viscosity experiments established a diffusional step as being significantly rate-limiting. Further studies with stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a combination of a number of physical and chemical steps. Investigation of the NO complexes of TyrH by means of optical absorption, electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) techniques revealed the relative positions of the substrate and cofactor with respect to NO, an O2 mimic, and provided further insight into how the active site is tuned for catalytic reactivity upon substrate and cofactor binding.

Le vieillissement normal s’accompagne d’une diminution du contenu du cholestérol cérébral. Au contraire, une accumulation de cholestérol est associée aux processus toxiques dans plusieurs pathologies dégénératives (maladie d’Huntington, maladie de Parkinson, épilepsie, maladie de Niemann Pick de type C, maladie d’Alzheimer). De plus, les parallèles étroits existent entre la physiopathologie moléculaire de la maladie d’Alzheimer et celle de la maladie de Niemann Pick de type C, maladie de l’homéostasie du cholestérol. Ainsi on retrouve dans ces deux pathologies une hyperphosphorylation de la protéine Tau, associée à une augmentation des endosomes élargis et à la production de peptides A. L’ensemble de ces éléments évoque le rôle potentiel de la surcharge en cholestérol cérébral comme facteur favorisant le développement de la maladie d’Alzheimer. L’objectif de mon travail de doctorat a été de déterminer si une surcharge en cholestérol in vivo dans les neurones de l’hippocampe, région précocement touchée par la maladie d’Alzheimer, pouvait être à l’origine de processus neurotoxiques et de modifications biochimiques et neuropathologiques proches de ceux qui sont observés dans cette pathologie. La quasi-totalité du cholestérol cérébral est synthétisée in situ, la barrière hémato-encéphalique ne permettant qu’un apport minime du cholestérol périphérique. L’excès de cholestérol est exporté de la circulation sanguine sous la forme du 24-hydroxycholestérol, un métabolite produit exclusivement dans les neurones par la cholestérol-24-hydroxylase codée par le gène Cyp46a1.La surcharge en cholestérol a été induite in vivo par inhibition de la cholestérol-24-hydroxylase, dans l’hippocampe par une stratégie d’ARN interférence délivré par une injection stéréotaxique d’un vecteur AAV5. Nous avons étudié, d’une part la capacité d’une accumulation de cholestérol à induire chez la souris normale, un phénotype clinique et neuropathologique proche de la maladie d’Alzheimer et d’autre part si cette même accumulation de cholestérol neuronal pouvait aggraver ou compléter le phénotype Alzheimer d’un modèle murin de la maladie, la souris APP23. L’injection du vecteur AAV5-shCYP46A1 dans la stratum lacunosum moleculare de l’hippocampe conduit à une inhibition significative de l'expression du gène Cyp46a1, associée à une diminution de la concentration du 24-hydroxycholestérol et une augmentation du contenu en cholestérol dans les neurones de l’hippocampe, 3 semaines après l’injection. En réponse à cet excès de cholestérol, des mécanismes régulateurs permettent de diminuer, d’une part l’import et d’augmenter l’export du cholestérol de la cellule et d’autre part d’augmenter le contenu en phosphatidylcholine afin de rétablir un ratio phospholipide/cholestérol physiologique. Cependant, l'accumulation majeure de cholestérol intracellulaire conduit, 3 semaines après l’injection, à une activation de la réponse UPR (Unfolded Protein Response ou stress du réticulum endoplasmique) caractérisée par l'expression des gènes codant les facteurs XBP1s, ATF6, GRP78 associée à celles des protéines PERK phosphorylée, CHOP et caspase 12, entraînant l'activation des caspases 9 et 3. Elle est associée à la phosphorylation des protéines GSK3 (Tyr216) et Tau (Thr231). En parallèle, l’augmentation du cholestérol induit, 3 semaines après l’injection, une augmentation de l’expression de la protéine Rab5 (marqueur des endosomes précoces) et une relocalisation de la protéine APP dans les fractions de radeaux lipidiques associées à l'activation de la voie amyloïdogénique (production des fragments-CTF et des peptides A42). L’étude lipidomique met en évidence, 4 semaines après l’injection, une augmentation du contenu en céramide à longues chaînes et à une augmentation des gangliosides. Tous ces éléments aboutissent à un processus de perte neuronale associée à un recrutement des astrocytes dès la quatrième semaine après l’injection.... / An increasing number of arguments suggest a close and complex link between cholesterol metabolism and neurodegenerative diseases, particularly with Alzheimer’s disease. Normal ageing is associated with a decrease of brain cholesterol content. Conversly, accumulation of brain cholesterol is associated with several neurodegenerative diseases (Huntington disease, Parkinson disease, epilepsy, Niemann Pick C disease, Alzheimer’s disease). Moreover, close connections exist between molecular physiopathology of AD and that of Niemann Pick, a disease of cholesterol homeostasis. Altogether, these results suggest that cholesterol overload might play a role, as an initiating factor for the development of AD.In the brain, cholesterol metabolism is tightly controlled. In adults, cholesterol is mainly synthetized by astrocytes, then shuttles to neurons where it is used. All cholesterol excess must be eliminated. Cholesterol cannot cross freely the blood-brain-barrier. To be metabolized, brain cholesterol must be converted in 24-hydroxy-cholesterol by the cholesterol-24 hydroxylase enzyme, coded by CYP46A1 gene. The objective of my PhD project was to determine if cholesterol accumulation in vivo in hippocampal neurons, a region early involved in AD pathology, could trigger neurotoxic processes with biochemical and neuropathological modifications close to what is observed in AD. Cholesterol overload in vivo was induced by inhibiting cholesterol 24-hydroxylase enzyme activity, using an RNA interference strategy. Stereotactic injection of an AAV5- shCYP46A1 vector in the stratum lacunosum moleculare of the hippocampus led to significant and rapid (as soon as 3 weeks after injection) inhibition of the Cyp46a1 gene in the hippocampus with an absence of RNA interference off-target effect. This inhibition was associated with a decrease of 24-hydroxycholesterol content and an increase of the cholesterol content. In response to this cholesterol excess, cell control mechanisms were initiated leading to decrease import and increase export of cholesterol, accompanied with an increase of phosphatidylcholine content to restore a physiological ratio of phospholipide/cholesterol. However, major accumulation of cholesterol led to neuronal death with activation of caspases 9 et 3, suggesting an apoptotic process. The cholesterol overload drives to an endoplasmic reticulum stress, with activation of the unfolded protein response (UPR) and expression of spliced XBP1, ATF6, GRP78, phosphorylated PERK, CHOP and caspase 12. These modifications were associated with phosphorylation of GSK3 (Tyr 216) and tau (Thr 231) proteins. In parallel, cholesterol accumulation led to increased expression of Rab5 (early endosome marker) and relocalization of APP in rafts domains associated to activation of amyloid pathway (production of -CTF fragments and A42 peptides). Lipidomic analysis showed an increase of ceramides and gangliosides content. All these modifications were associated with neuronal death 4 weeks after injection and astrocytosis, leading to an EEG theta rhythm accelerated to beta frequencies, memory deficits and hippocampal atrophy. In a mouse model of Alzheimer disease, the APP23 mouse, cholesterol accumulation led to major aggravation of the phenotype, with increased production of A peptides, occurring of tau phosphorylation and UPR response, leading to accelerated neuronal death. Altogether, these results suggest a direct link between cholesterol accumulation in the brain and Alzheimer’s disease. Brain cholesterol accumulation could seed the sows to the development of Alzheimer’s pathology. Reducing cerebral cholesterol could thus be a relevant therapeutic strategy to prevent the development, or at least slow down the evolution of the pathology in Alzheimer’s disease.

La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l’étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l’obstacle majeur à l’exploitation industrielle de la biomasse lignocellulosique. L’acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d’identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l’implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l’isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d’acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l’isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d’acide p-coumarique, une baisse du rapport S/G ainsi que l’accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d’un quart par rapport au sauvage. / Brachypodium distachyon was recently adopted as an experimental model for grass species. As such, it is used to study grass cell wall, in particular in the context of their use as renewable feedstock for the production of second generation bioethanol. Lignins are polymers of three main units (H, G and S) originating from the polymerization of monolignols, and are linked to hydroxycinnamic acids in grasses. They constitute the main bottleneck to industrial processes targeting lignocellulosic biomass and improving the understanding of the mechanisms directing their structure and deposition could lead to the identification of the factors modulating associated production yields. Four gene families were studied and the involvement of three genes in the monolignols biosynthetic pathway was shown: BdF5H2 displays a ferulate-5-hydroxylase activity enabling the synthesis of the S lignin units, BdCOMT3 is the main caffeic acid O-methyltransferase and its partial loss of function in two independent mutant lines leads to the reduction of lignin content, the modification of the S/G units ratio and a decrease in p-coumaric acid accumulation. BdCAD1 is the main cinnamyl alcohol dehydrogenase isoform: its loss of function in two independent mutant lines results in a decrease in lignin content and of the S/G ratio and the accumulation of sinapaldehyde. Moreover, these two lines display significatively increased saccharification yields.

Verticillium longisporum ist ein bodenbürtiger, phytopathogener Pilz, der Pflanzen der Familie der Brassicaceen befällt. Er dringt durch die Wurzel ein und verbreitet sich in der Pflanze über das Xylem. In dieser Arbeit wurden die metabolischen Veränderungen in der Modellpflanze Arabidopsis thaliana während der Pflanzen-Pilz-Interaktion analysiert. Hierfür wurde die

Ziel der Untersuchungen war, zur Klärung der Ursachen einer während der letzten Jahrzehnte erhöhten Frequenz sowohl von PCOS als auch von IO beizutragen. Es war zu ermitteln, ob hormonelle Verschiebungen bei den Patienten nachweisbar und diese durch genetische und epigenetische Faktoren erklärbar sind. Ausgehend von dem Postulat, daß verminderte 21-OH- und 3beta-HSD-Aktivitäten als prädisponierende Faktoren von PCOS und IO angesehen werden, waren hormonanalytische Untersuchungen zur Ermittlung partieller 21-OH- bzw. 3beta-HSD-Defizienzen durchgeführt worden. Den eigenen Erfahrungen und Darstellungen der internationalen Literatur entsprechend befaßt sich ein Teil der Methodik mit der Entwicklung einer neuen, der üblichen 17alfa-OHP-Messung überlegenen Methode zur Ermittlung von 21-OH-Defizienzen durch 21-DOF-Bestimmung nach ACTH-Test im Blutplasma. Wir erhielten bei vier von 21 PCOS-Patientinnen und drei von acht Patienten mit IO erhöhte 21-DOF-, 21-DOF/F- bzw. 17alfa-OHP-Werte nach ACTH-Test, die auf partielle 21-OH-Defizienzen hinweisen. Zusätzlich wurden bei 12 PCOS-Patientinnen erhöhte basale DHEAS- oder DHEAS/F-Werte gefunden, die als Hinweise auf partielle 3beta-HSD-Defizienzen oder 17,20-Lyase-Hyperaktivität gedeutet wurden. In der Stichprobe der IO waren DHEAS oder DHEAS/F-Werte bei vier Patienten erhöht. Da bei vier der 12 Patientinnen mit PCOS und zwei von vier Patienten mit IO genetisch und endokrinologisch gleichzeitig eine partielle 21-OH-Defizienz nachgewiesen wurde, kann bei diesen Patienten eine partielle 3beta-HSD-Defizienz weitgehend ausgeschlossen werden. Es wurden molekulargenetische Untersuchungen für die 14 häufigsten Mutationen in CYP21 bei Cohorten mit AGS, PCOS und IO durchgeführt. Die Untersuchung der AGS-Patienten sollte dazu dienen, ein effizientes und schnelles System der Mutationssuche für diagnostische Zwecke zu etablieren. Es wurden die häufigsten, phänotypisch wirksamen Mutationen in CYP21 bei der Mehrzahl dieser Patientengruppe im homozygoten bzw. compound heterozygoten Zustand gefunden und eine deutliche Genotyp-Phänotyp-Korrelation festgestellt. Auch bei Patientinnen mit PCOS sowie bei IO, bei denen partielle 21-OH-Defizienzen nachweisbar waren, wurden Mutationen in CYP21 gefunden. Die hierbei heterozygot vorliegenden Mutationen waren dieselben, die homozygot oder compound heterozygot bei schweren Formen des AGS gefunden wurden. Es ergab sich eine Korrelation molekulargenetischer und hormonanalytischer Befunde bei AGS, PCOS sowie IO. Allerdings konnten bei der Mehrzahl der Fälle mit PCOS und mit IO weder Mutationen noch hormonelle Auffälligkeiten hinsichtlich partieller 21-OH-Defizienzen gefunden werden. Die jedoch bei vielen Patientinnen gefundenen erhöhten DHEAS- und DHEAS/F-Werte stimmen mit Untersuchungen überein, die parallel starke Zunahmen der Häufigkeit der Hemmung des Enzyms 3ß-HSD bzw. der Aktivierung der 17,20-Lyase bei PCOS-Patientinnen und der Prävalenz des PCOS selbst bei nach 1955 geborenen Frauen und von Spermatogenesestörungen bei nach 1960 geborenen Männern fanden. Die Ursache hierfür wird in der Beeinflussung der adrenalen und gonadalen Steroidhormonsynthese vor allem durch das Umweltteratogen DDT und seine Metaboliten gesehen. Weiterhin wurde der Umweltfaktor Streß diskutiert. Für die Ätiopathogenese der untersuchten Fertilitätsstörungen werden materno-fetale Mechanismen postuliert, worauf unsere sowohl molekulargenetischen als auch hormonanalytischen Befunde hinweisen. Insgesamt bestätigen die Ergebnisse unserer Arbeit die These, daß Leben auf der Interaktion von Genen und Umweltfaktoren beruht und daß Hormone dabei als Mediatoren wirken. In gen- oder umweltbedingten unphysiologischen Konzentrationen können sie während kritischer Entwicklungsphasen des neuroendokrinen Systems als Teratogene wirken und zu lebenslangen Reproduktionsstörungen führen. / This paper describes a mutational and hormonal screening in a cohort of 21 patients ultrasonically diagnosed with PCO. Our data show single heterozygous base pair CYP21 mutations in 4 patients. The four women with PCOS and CYP21 mutations also displayed clear signs of partial 21-hydroxylase deficiency through a significant rise in 21DOF or 17alfa-OHP plasma levels after ACTH stimulation. Azziz et al. have reported several heterozygous mutations in hyperandrogenic women with LO-CAH. Other studies report several heterozygous point mutations in hyperandrogenic woman who, however, were not examined for polycystic ovaries.The correlation between the hormone profiles and genetic screening results found with our patients underscores the latter s usefulness with PCOS patients. In contrast to the hormone profile, genetic screening is not influenced by external factors. The frequency of heterozygous CYP21 mutations is higher (19%) than in the normal population (5-8%), suggesting a link with PCOS in some cases. The ratio of LH/FSH was significantly raised in 43% of the cases. Most importantly, basal plasma DHEA-S levels and DHEA-S/F ratios were clearly increased, higher than the means +2SD in controls. This suggests a partial 3beta-hydroxysteroid dehydrogenase deficiency or 17,20 lyase hyperactivity. Other authors, however, were not able to find mutations in the corresponding genes. This could be explained by the fact that the DDT metabolite o,p DDD is a strong inhibitor of 3beta-HSD, and that DDT and its metabolites may be able to activate the 17,20 lyase, a cytochrome P450 enzyme. Furthermore, DDT has some oestrogen activity, and its perinatal administration can produce a PCOS-like syndrome in rats. Very significantly, there has not only been an approximately fourfold increased prevalence of PCO in women borne since 1955 in eastern Germany, following a massive prenatal exposure to DDT, but also a notable shift in the hormone profiles of those affected. A predominance of 3beta-HSD deficiencies and 17,20 lyase hyperactivity (70%) vs. 21-hydroxylase deficiency (23%) has emerged, in contrast with 21-hydroxylase deficiencies in 70% vs. 3beta-HSD deficiencies or 17,20 lyase hyperactivity in 14% for those born earlier than 1955. Similar results were obtained in this study for women with PCOS born since 1955, suggesting that the prenatal exposure of high amounts of DDT and its metabolites indeed appear to be responsible - at least in part - for the major increase in PCO and PCOS.

Estudos apontam a neuroinflamação como um dos possíveis agravantes do mal de Parkinson. Aqui investigamos a susceptibilidade de duas linhagens de camundongos selecionados quanto a suas capacidades de produzir alta (AIRmax) ou baixa (AIRmin) resposta inflamatória aguda aos agentes neurotóxicos 1-Metil-4-Fenil-1,2,3,6-Tetrahidropirimidina (MPTP) (Tratamento agudo: 4 x 20 mg/kg ou 5 x 20 mg/kg s.c. a cada 2 horas em um único dia) e rotenona (tratamento crônico: infusão s.c. contínua por bomba osmótica Alzet a 3, 6 e 12 mg/kg/dia por 28 dias ou sub-crônico: injeção i.p na dose de 3 mg/kg/dia durante 10 dias). A avaliação motora contou com Rotarod empregando paradigma de 5 minutos e rotação crescente de 5 a 50 rpm. A lesão foi quantificada por imunohistoquímica para tirosina hidroxilase em cortes de estriado e substância negra. Os resultados sugerem que ambas as linhagens AIRmax e AIRmin, são resistentes a lesão neuronal por MPTP ou rotenona. Conjecturamos que as linhagens se diferenciam quanto a resposta inflamatória periférica, mas não de origem central (neuroinflamação). / Studies suggest that neuroinflammatory processes are involved in Parkinson\'s disease. Here we investigated the susceptibility of two inbred strains of mice selected for their ability to produce high (AIRmax) or low (AIRmin) acute inflammatory response to neurotoxins 1-Methyl-4-phenyl-1,2,3,6-Tetrahydropyridine (MPTP) (4 x 20 mg/kg or 5 x 20 mg/kg s.c. every 2 hours in a single day) and rotenone (chronic treatment: continuous s.c. infusion of 3, 6 and 12 mg/kg/day by Alzet osmotic pump for 28 days or sub-chronic treatment: i.p. injection at a dose of 3 mg/kg/day for 10 days). The animals were evaluated by rotarod apparatus in sessions of 5 minutes and constantly increasing speed from 5 to 50 rpm. The lesion was quantified by tyrosine hydroxylase immunohistochemistry in sections of striatum and substantia nigra. The results suggest that both strains AIRmax and AIRmin are resistant to neuronal damage by MPTP or rotenone. We conjecture that the strains differ concerning the peripheral inflammatory response, but not in neuroinflammatory mechanisms.

Investigamos os efeitos do treinamento físico (T) sobre a densidade da tirosina hidroxilase (TH) em arteríolas e adrenais (respostas neuro-hormonais). SHR machos foram treinados (55% da capacidade máxima) ou mantidos sedentários (S). Após o registro da PA e FC basais, foram anestesiados para obtenção dos tecidos (músculos locomotores: sóleo, gastrocnêmio, grácil; não-locomotor: temporal; rins e supra-renais) para o Western Blot ou imunohistoquímica. O T aumentou a capacidade física (+77%) e reduziu PA e FC (-6% e -10%, p<0.05). Houve aumento da TH imunorreatividade nos músculos locomotores (+57%, p<0.05), com aumento discreto no temporal (+24%, p>0.05), sem alteração nos rins. Houve normalização da razão parede/luz apenas nas arteríolas de músculos locomotores, sem alteração da TH nas supra-renais. O T aumenta a síntese/armazenamento da noradrenalina nas arteríolas musculares esqueléticas (resposta neural), uma compensação à redução da razão p/l para manutenção do fluxo sanguíneo local. A não alteração de TH no temporal sugere que a resposta neural é modulada por fatores locais / We investigated the effects of training (T) on tyrosine hydroxylase (TH) density in arterioles and adrenals (neuro-hormoral responses). Male SHR were submitted to treadmill T (55% of maximal capacity) or kept sedentary (S). After AP and HR recordings, rats were anesthetized and tissues (soleus, gastrocnemius red, gracilis = locomotor; temporalis = non-locomotor muscle; kidney and adrenals) collected for Western Blot and immunohistochemistry. T improved treadmill performance (+77%) and reduced AP and HR (-6% and -10%, p<0.05). TH immunoreactivity was increased in locomotor muscles (+57%, p<0.05) with smaller changes on temporalis (+24%, p>0.05) without any change on kidneys. Enlarged arterioles wall/lumen ratio was reduced only in locomotor muscles; there was no change on adrenals TH content. T increases noradrenaline synthesis/storage on skeletal muscles arterioles, which represents a compensatory neural response to T-induced structural remodeling in order to maintain a near normal local flow. Absence of changes on temporalis TH suggests that neural response is modulated by local factors

O objetivo central do presente estudo foi avaliar se os agonistas adrenérgicos a2 e imidazólicos, importantes drogas de ação anti-hipertensiva utilizadas na clínica médica, podem atuar também na região comissural do núcleo do trato solitário (NTSc), o qual constituí uma importante região do bulbo envolvida no controle cardiovascular. Em ratos Wistar adultos, observamos que a hipotensão produzida pela injeção de moxonidina no 4º V foi reduzida após a lesão eletrolítica do NTSc. Ademais, a injeção de moxonidina no NTSc reduziu a pressão arterial média (PAM), a frequência cardíaca (FC) e a atividade simpática (AS). A injeção de antagonistas adrenérgicos (ioimbina ou RX821002) no NTSc foi capaz de bloquear as respostas hipotensora e de simpatoinibição produzida pela moxonidina no NTSc. A injeção bilateral de moxonidina na região RVL/C1 reduziu PAM e AS de maneira mais intensa do que as injeções no NTSc. Em concordância com os resultados apresentados, mostramos que a atividade elétrica dos neurônios da região do RVL/C1 foi reduzida após a injeção de moxonidina no NTSc. Concluímos que a moxonidina pode produzir os seus efeitos anti-hipertensivos atuando também sobre o NTSc. / The main objective of this study was to evaluate whether the a2 adrenergic and imidazoline agonists, important antihypertensive drugs used in clinical medicine, may also act in the commissural region of the nucleus of the solitary tract (cNTS), which constitutes an important region of brainstem involved in cardiovascular control. In adult rats, the hypotension elicited by central injections of moxonidine was reduced after electrolytic lesion of cNTS. Furthermore, injection of moxonidine into the cNTS reduced mean arterial pressure (MAP), heart rate (HR) and sympathetic activity (SNA). Injection of the a2 adrenergic antagonist (RX821002 or yohimbine) into the cNTS completely blocked the hypotension and sympathoinhibition responses produced by moxonidine into the cNTS. Bilateral injection of moxonidine in the RVLM/C1 produced huge effects on MAP and SNA in comparison of cNTS injections. In agreement with our results, moxonidine-injected into the cNTS also elicited a reduction in the activity of RVLM/C1 neurons. Our conclusion is that moxonidine may produce their antihypertensive effects also acting on cNTS neurons.