We hypothesized that, due to the high pH of this compartment, the reticulum epithelium displays particular features in the transport of short-chain fatty acids (SCFA). Ovine reticulum epithelium was incubated in Ussing chambers using a bicarbonate-free buffer solution containing butyrate (20 mmol L−1). p-hydroxymercuribenzoic acid (pHMB), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), or ouabain were added to the buffer solution as inhibitors of monocarboxylate transporters, sodium-proton-exchangers, or the Na+/K+-ATPase, respectively. The short-circuit current (Isc) and transepithelial conductance (Gt) were monitored continuously while the flux rates of 14C-labelled butyrate were measured in the mucosal-to-serosal (Jmsbut) or serosal-to-mucosal direction (Jsmbut). Under control conditions, the mean values of Isc and Gt amounted to 2.54 ± 0.46 µEq cm−2 h−1 and 6.02 ± 3.3 mS cm−2, respectively. Jmsbut was 2.1 ± 1.01 µmol cm−2 h−1 on average and about twice as high as Jsmbut. Incubation with ouabain reduced Jmsbut, while Jsmbut was not affected. The serosal addition of EIPA did not affect Jmsbut but reduced Jsmbut by about 10%. The addition of pHMB to the mucosal or serosal solution reduced Jmsbut but had no effect on Jsmbut. Mucosally applied pHMB provoked a transient increase in the Isc. The serosal pHMB sharply reduced Isc. Our results demonstrate that butyrate can be effectively transported across the reticulum epithelium. The mechanisms involved in this absorption differ from those known from the rumen epithelium.

No description available.

La grossesse s’accompagne d’importantes modifications hormonales et hémodynamiques. Parmi celles-ci, le système rénine-angiotensine-aldostérone (SRAA) est activé très tôt durant la grossesse. De plus, cette augmentation du SRAA est accompagnée d’élévations du débit cardiaque et du volume plasmatique ainsi que des baisses paradoxales de la pression artérielle et de la résistance vasculaire périphérique. Ceci suggère que la grossesse induit un remaniement des réponses physiologiques normales au SRAA. Une résistance vasculaire à l’action des vasopresseurs est également observée durant la gestation. Ce phénomène serait causé par la modification de la fonction des canaux calciques et potassiques. De plus, il serait possiblement dû à la participation de la Na+/K+-ATPase, par son influence sur le potentiel membranaire des cellules des muscles lisses vasculaires (VSMC). La présence des récepteurs minéralocorticoïdes (MR) dans les VSMC laisse croire que l’aldostérone peut influencer le tonus vasculaire par des effets génomiques et non-génomiques. Compte tenu des connaissances actuelles, nous avons émis l’hypothèse que l’augmentation des taux sériques d’aldostérone durant la grossesse est responsable des changements hémodynamiques observés et que ces effets sont causés par l’activation des MR. Des rates gestantes ont été traitées avec du canrénoate de potassium (20 mg/kg•jr), un antagoniste des MR, durant la dernière semaine de gestation (sur 3). Sur des anneaux aortiques dénudés de leur endothélium, nous avons mesuré les réponses contractiles à la phényléphrine (PhE) et au KCl en présence d’un bloqueur des canaux calciques dépendants du voltage (VDCC), la nifédipine, et d’activateurs des canaux potassiques à large conductance (BKCa) et ceux dépendants de l’ATP (KATP), respectivement le NS-1619 et la cromakalim. Les réponses à la PhE et au KCl sont réduites à partir du 17e jour de gestation et le traitement au canrénoate augmente ces réponses dans tous les groupes. Les modulateurs de canaux ioniques atténuent les réponses à la PhE et au KCl. Cependant, le canrénoate modifie aussi les effets des modulateurs sur les aortes. Aucun effet ou une baisse des réponses est observable chez les rates non gestantes, tandis qu’une hausse de leur effet inhibiteur est notée chez les rates gestantes. Ces effets du canrénoate font croire que l’aldostérone participe à l’adaptation de la réactivité vasculaire durant la grossesse. Par ailleurs, le potentiel membranaire des VSMC pourrait être affecté dans la gestation. Pour vérifier cette hypothèse, nous avons évalué l’activité de la Na+/K+-ATPase, impliquée dans le contrôle du potentiel membranaire. Nos résultats démontrent que l’activité de la pompe est inhibée à partir du 19e jour de gestation. Cet effet est renversé par le canrénoate. Toutefois, comme le renversement de l’inhibition de la pompe est également présent chez les rates gestantes traitées avec du PST 2238, un antagoniste de l’ouabaïne sur la Na+/K+-ATPase, et que le canrénoate agit également comme agoniste partiel de la pompe, nous croyons que la diminution d’activité associée à la gestation est liée à une inhibition de la Na+/K+-ATPase par des stéroïdes cardiotoniques plutôt qu’à un effet des minéralocorticoïdes. L’augmention d’activité de la pompe liée au canrénoate s’accompagne d’une diminution de l’expression de la sous-unité α1, suggérant que la sous-unité α2 est responsable des variations de contractilité de l’aorte, puisque son expression n’est pas modifiée par le canrénoate. Les effets de la diminution de l’expression de la sous-unité α1, influencée par la signalisation du MR, restent à être déterminés. Néanmoins, nos résultats montrent que les modifications d’activité de la Na+/K+-ATPase influencent l’activité des canaux potassiques et que la pompe pourraient être un des éléments primordiaux dans le contrôle de la réactivité vasculaire durant la grossesse. Comme le canrénoate modifie la réactivité vasculaire, nous voulions déterminer ses impacts sur la pression artérielle. Des rates gestantes ont été traitées avec du canrénoate (20 ou 60 mg/kg•jr) et les paramètres hémodynamiques ont été évalués par radiotélémétrie. Aucune modification de la pression artérielle, du rythme cardiaque et de la pression pulsée ne sont mesurées chez les rates recevant le traitement. Toutefois, des augmentations de l’osmolalité, des taux sériques d’aldostérone et de corticostérone ainsi que de l’activité rénine plasmatique sont observées chez les animaux recevant 60 mg/kg•jr. Le canrénoate bloque donc le rétrocontrôle du SRAA. Par contre, les MR ne sont pas les principaux responsables du contrôle de la pression artérielle durant la grossesse. En conclusion, nous avons démontré que le traitement des rates au canrénoate influence la réactivité vasculaire de l’aorte durant la gestation. Cet effet est causé par la modification de l’activité de certains canaux ioniques (VDCC, BKCa et KATP). De plus, le canrénoate renverse l’inhibition de la Na+/K+-ATPase observée durant la gestation. Finalement, les actions locales de cet antagoniste des MR sur les vaisseaux sanguins ne se répercutent pas sur l’effet systémique global et aucune modification de la pression artérielle n’est observée. D’autres études seront toutefois nécessaires pour déterminer les voies de signalisation par lesquelles l’aldostérone module les réponses des canaux ioniques dans les VSMC. / Pregnancy is accompanied by important hormonal and hemodynamic modifications. Among them, the renin-angiotensin-aldosterone system (RAAS) is activated early during pregnancy. Furthermore, this increase of RAAS is accompanied by raises of cardiac output and blood volume as well as paradoxal decreases in blood pressure and peripheral vascular resistance. These suggest that pregnancy induces reorganization of the normal physiological responses to RAAS. In addition, a decreased vascular reactivity to vasoconstrictive agents is observed during pregnancy. Modifications of calcium and potassium channels function would be implicated in this phenomenon. Furthermore, an implication of the Na+/K+-ATPase is suspected, through its influence on the membrane potential of vascular smooth muscle cells (VSMC). Aldosterone, through the presence of mineralocorticoid receptors (MR) in VSMC, could control vascular tone by its genomic and non-genomic effects. With our knowledge at this time, we submit the hypothesis that the increased serum aldosterone levels of pregnancy are responsible for the hemodynamic changes associated with pregnancy and that these effects are caused by the activation of MR. Pregnant rats were treated with potassium canrenoate (20 mg/kg•d), a MR antagonist, during the last week of pregnancy (out of 3 weeks). Vascular reactivity of endothelium-denuded aortic rings was measured. Contractile responses to phenylephrine (PhE) and KCl were studied in the presence of a voltage-dependent calcium channel (VDCC) blocker, nifedipine, as well as calcium-actived (BKCa) and ATP-dependent (KATP) potassium channels activators, NS-1619 and cromakalim respectively. Vascular responses to PhE and KCl were reduced as of the 17th day of gestation. Canrenoate increased the responses to both agonists in the aortas of all the groups. Ionic channel modulators reduced the contractile responses to PhE and KCl. However, canrenoate also modified the responses to the modulators. In the aortas of non pregnant rats, no effect or a decrease of their inhibitory effect were observed while with the pregnant rats we noticed an increased effect. These results suggest that aldosterone could be implicated in the adaptation of vascular responses to pregnancy. On the other hand, VSMC membrane potential could be affected during pregnancy. To verify this hypothesis, Na+/K+-ATPase activity was evaluated since it is implicated in its control. Our results demonstrated that the pump is inhibited as of the 19th day of pregnancy. This effect was reversed by canrenoate. However, since the reversal of the inhibition of the pump was also present in pregnant rats treated with PST 2238, an ouabain antagonist on the Na+/K+-ATPase, and because canrenoate can also act as a partial agonist of the pump, we believe that the decreased activity associated with pregnancy is linked to the inhibition of the Na+/K+-ATPase by cardiotonic steroids rather than the effect of mineralocorticoids. The increased activity of the pump by canrenoate is linked with a decreased of the α1 subunit expression. This suggests that the α2 subunit of the pump would be responsible for the variations of aortic contractility since its expression is not modified by canrénoate. The effects of the diminished expression of the α1 subunit, influenced by the MR signalization, still need to be determined. Nevertheless, our results showed that modifications of Na+/K+-ATPase activity had an impact on the activity of potassium channels and that the pump could be one of the principal elements implicated in the control of vascular reactivity during pregnancy. Since canrenoate modified vascular reactivity, we wanted to determine its impact on blood pressure. Pregnant rats were treated with canrenoate (20 or 60 mg/kg•d) and hemodynamic functions were determined by radiotelemetry. No modification was observed in blood pressure, cardiac output and pulse pressure among the treated rats. However, increases in osmolality, aldosterone and corticosterone levels as well as plasma renin activity were observed in the animal receiving the 60 mg/kg•d canrénoate. Thus, canrenoate blocks the negative feedback of the RAAS. However, MR are not the principal element responsible for the control of blood pressure during pregnancy. In conclusion, we have demonstrated that canrenoate affects aortic vascular reactivity during pregnancy. This effect is perpetuated by modifying the activity of certain ionic channels (VDCC, BKCa and KATP). Furthermore, canrenoate is able to reverse the inhibition of the Na+/K+-ATPase observed during pregnancy. Finally, the local actions of this MR antagonist on blood vessels are not reflected by the global systemic effect and no modification of the blood pressure was observed. Other studies will be necessary to determine the signaling pathways by which aldosterone modulate ionic channel responses in VSMC.

La grossesse s’accompagne d’importantes modifications hormonales et hémodynamiques. Parmi celles-ci, le système rénine-angiotensine-aldostérone (SRAA) est activé très tôt durant la grossesse. De plus, cette augmentation du SRAA est accompagnée d’élévations du débit cardiaque et du volume plasmatique ainsi que des baisses paradoxales de la pression artérielle et de la résistance vasculaire périphérique. Ceci suggère que la grossesse induit un remaniement des réponses physiologiques normales au SRAA. Une résistance vasculaire à l’action des vasopresseurs est également observée durant la gestation. Ce phénomène serait causé par la modification de la fonction des canaux calciques et potassiques. De plus, il serait possiblement dû à la participation de la Na+/K+-ATPase, par son influence sur le potentiel membranaire des cellules des muscles lisses vasculaires (VSMC). La présence des récepteurs minéralocorticoïdes (MR) dans les VSMC laisse croire que l’aldostérone peut influencer le tonus vasculaire par des effets génomiques et non-génomiques. Compte tenu des connaissances actuelles, nous avons émis l’hypothèse que l’augmentation des taux sériques d’aldostérone durant la grossesse est responsable des changements hémodynamiques observés et que ces effets sont causés par l’activation des MR. Des rates gestantes ont été traitées avec du canrénoate de potassium (20 mg/kg•jr), un antagoniste des MR, durant la dernière semaine de gestation (sur 3). Sur des anneaux aortiques dénudés de leur endothélium, nous avons mesuré les réponses contractiles à la phényléphrine (PhE) et au KCl en présence d’un bloqueur des canaux calciques dépendants du voltage (VDCC), la nifédipine, et d’activateurs des canaux potassiques à large conductance (BKCa) et ceux dépendants de l’ATP (KATP), respectivement le NS-1619 et la cromakalim. Les réponses à la PhE et au KCl sont réduites à partir du 17e jour de gestation et le traitement au canrénoate augmente ces réponses dans tous les groupes. Les modulateurs de canaux ioniques atténuent les réponses à la PhE et au KCl. Cependant, le canrénoate modifie aussi les effets des modulateurs sur les aortes. Aucun effet ou une baisse des réponses est observable chez les rates non gestantes, tandis qu’une hausse de leur effet inhibiteur est notée chez les rates gestantes. Ces effets du canrénoate font croire que l’aldostérone participe à l’adaptation de la réactivité vasculaire durant la grossesse. Par ailleurs, le potentiel membranaire des VSMC pourrait être affecté dans la gestation. Pour vérifier cette hypothèse, nous avons évalué l’activité de la Na+/K+-ATPase, impliquée dans le contrôle du potentiel membranaire. Nos résultats démontrent que l’activité de la pompe est inhibée à partir du 19e jour de gestation. Cet effet est renversé par le canrénoate. Toutefois, comme le renversement de l’inhibition de la pompe est également présent chez les rates gestantes traitées avec du PST 2238, un antagoniste de l’ouabaïne sur la Na+/K+-ATPase, et que le canrénoate agit également comme agoniste partiel de la pompe, nous croyons que la diminution d’activité associée à la gestation est liée à une inhibition de la Na+/K+-ATPase par des stéroïdes cardiotoniques plutôt qu’à un effet des minéralocorticoïdes. L’augmention d’activité de la pompe liée au canrénoate s’accompagne d’une diminution de l’expression de la sous-unité α1, suggérant que la sous-unité α2 est responsable des variations de contractilité de l’aorte, puisque son expression n’est pas modifiée par le canrénoate. Les effets de la diminution de l’expression de la sous-unité α1, influencée par la signalisation du MR, restent à être déterminés. Néanmoins, nos résultats montrent que les modifications d’activité de la Na+/K+-ATPase influencent l’activité des canaux potassiques et que la pompe pourraient être un des éléments primordiaux dans le contrôle de la réactivité vasculaire durant la grossesse. Comme le canrénoate modifie la réactivité vasculaire, nous voulions déterminer ses impacts sur la pression artérielle. Des rates gestantes ont été traitées avec du canrénoate (20 ou 60 mg/kg•jr) et les paramètres hémodynamiques ont été évalués par radiotélémétrie. Aucune modification de la pression artérielle, du rythme cardiaque et de la pression pulsée ne sont mesurées chez les rates recevant le traitement. Toutefois, des augmentations de l’osmolalité, des taux sériques d’aldostérone et de corticostérone ainsi que de l’activité rénine plasmatique sont observées chez les animaux recevant 60 mg/kg•jr. Le canrénoate bloque donc le rétrocontrôle du SRAA. Par contre, les MR ne sont pas les principaux responsables du contrôle de la pression artérielle durant la grossesse. En conclusion, nous avons démontré que le traitement des rates au canrénoate influence la réactivité vasculaire de l’aorte durant la gestation. Cet effet est causé par la modification de l’activité de certains canaux ioniques (VDCC, BKCa et KATP). De plus, le canrénoate renverse l’inhibition de la Na+/K+-ATPase observée durant la gestation. Finalement, les actions locales de cet antagoniste des MR sur les vaisseaux sanguins ne se répercutent pas sur l’effet systémique global et aucune modification de la pression artérielle n’est observée. D’autres études seront toutefois nécessaires pour déterminer les voies de signalisation par lesquelles l’aldostérone module les réponses des canaux ioniques dans les VSMC. / Pregnancy is accompanied by important hormonal and hemodynamic modifications. Among them, the renin-angiotensin-aldosterone system (RAAS) is activated early during pregnancy. Furthermore, this increase of RAAS is accompanied by raises of cardiac output and blood volume as well as paradoxal decreases in blood pressure and peripheral vascular resistance. These suggest that pregnancy induces reorganization of the normal physiological responses to RAAS. In addition, a decreased vascular reactivity to vasoconstrictive agents is observed during pregnancy. Modifications of calcium and potassium channels function would be implicated in this phenomenon. Furthermore, an implication of the Na+/K+-ATPase is suspected, through its influence on the membrane potential of vascular smooth muscle cells (VSMC). Aldosterone, through the presence of mineralocorticoid receptors (MR) in VSMC, could control vascular tone by its genomic and non-genomic effects. With our knowledge at this time, we submit the hypothesis that the increased serum aldosterone levels of pregnancy are responsible for the hemodynamic changes associated with pregnancy and that these effects are caused by the activation of MR. Pregnant rats were treated with potassium canrenoate (20 mg/kg•d), a MR antagonist, during the last week of pregnancy (out of 3 weeks). Vascular reactivity of endothelium-denuded aortic rings was measured. Contractile responses to phenylephrine (PhE) and KCl were studied in the presence of a voltage-dependent calcium channel (VDCC) blocker, nifedipine, as well as calcium-actived (BKCa) and ATP-dependent (KATP) potassium channels activators, NS-1619 and cromakalim respectively. Vascular responses to PhE and KCl were reduced as of the 17th day of gestation. Canrenoate increased the responses to both agonists in the aortas of all the groups. Ionic channel modulators reduced the contractile responses to PhE and KCl. However, canrenoate also modified the responses to the modulators. In the aortas of non pregnant rats, no effect or a decrease of their inhibitory effect were observed while with the pregnant rats we noticed an increased effect. These results suggest that aldosterone could be implicated in the adaptation of vascular responses to pregnancy. On the other hand, VSMC membrane potential could be affected during pregnancy. To verify this hypothesis, Na+/K+-ATPase activity was evaluated since it is implicated in its control. Our results demonstrated that the pump is inhibited as of the 19th day of pregnancy. This effect was reversed by canrenoate. However, since the reversal of the inhibition of the pump was also present in pregnant rats treated with PST 2238, an ouabain antagonist on the Na+/K+-ATPase, and because canrenoate can also act as a partial agonist of the pump, we believe that the decreased activity associated with pregnancy is linked to the inhibition of the Na+/K+-ATPase by cardiotonic steroids rather than the effect of mineralocorticoids. The increased activity of the pump by canrenoate is linked with a decreased of the α1 subunit expression. This suggests that the α2 subunit of the pump would be responsible for the variations of aortic contractility since its expression is not modified by canrénoate. The effects of the diminished expression of the α1 subunit, influenced by the MR signalization, still need to be determined. Nevertheless, our results showed that modifications of Na+/K+-ATPase activity had an impact on the activity of potassium channels and that the pump could be one of the principal elements implicated in the control of vascular reactivity during pregnancy. Since canrenoate modified vascular reactivity, we wanted to determine its impact on blood pressure. Pregnant rats were treated with canrenoate (20 or 60 mg/kg•d) and hemodynamic functions were determined by radiotelemetry. No modification was observed in blood pressure, cardiac output and pulse pressure among the treated rats. However, increases in osmolality, aldosterone and corticosterone levels as well as plasma renin activity were observed in the animal receiving the 60 mg/kg•d canrénoate. Thus, canrenoate blocks the negative feedback of the RAAS. However, MR are not the principal element responsible for the control of blood pressure during pregnancy. In conclusion, we have demonstrated that canrenoate affects aortic vascular reactivity during pregnancy. This effect is perpetuated by modifying the activity of certain ionic channels (VDCC, BKCa and KATP). Furthermore, canrenoate is able to reverse the inhibition of the Na+/K+-ATPase observed during pregnancy. Finally, the local actions of this MR antagonist on blood vessels are not reflected by the global systemic effect and no modification of the blood pressure was observed. Other studies will be necessary to determine the signaling pathways by which aldosterone modulate ionic channel responses in VSMC.

Monosialoganglioside (GM1) is a component of most cell membranes and is thought to play a role in development, recognition and cellular differentiation. Furthermore, GM1 is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. In the present study we investigate the effect of GM1 on the catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, on the spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cortex of rats ex vivo and in vitro. Systemic GM1 administration (50 mg/kg, i.p.; twice) reduced spontaneous chemiluminescence and increased CAT activity ex vivo. On the other hand, GM1 (103-104 nM) reduced CAT activity in vitro. The other parameters were not affected by GM1 administration. These findings agree with the view that the antioxidant action of GM1 is not due to an intrinsic antioxidant activity of this glycolipid, but due to a secondary decrease of reactive species generation and/or increase of antioxidant defenses. Moreover, we evaluated whether GM1 could have a neuroprotective action on the experimental model of glutaric acidemia, an inherited metabolic disorder characterized by glutaric acid (GA) accumulation and neurological dysfunction, as striatal degeneration and convulsion. The systemic GM1 administration (50 mg/kg, i.p. twice) protected against the convulsions, oxidative damage markers increase (total protein carbonylation and thiobarbituric acid-reactive substances - TBARS) production and Na+,K+-ATPase activity inhibition induced by GA (4 mol/ 2 l) in striatum of rats. Furthermore, convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum, but not with oxidative stress marker measures. In addition, GM1 (50-200 M) protected against Na+,K+-ATPase inhibition induced by GA (6 mM), but not against oxidative damage in vitro. Intrastriatal administration of muscimol (46 pmol/striatum), a GABAA receptor agonist, but not glutamatergic receptor antagonists MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum), prevented GA-induced convulsions and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance in the injected striatum with GA. Since GM1 and muscimol prevented neurotoxic effects induced by GA, we investigated the GM1 action after intrastriatal administration of pentylenetetrazole (PTZ), a GABAA receptor antagonist. GM1 treatment prevented seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl induced by PTZ (1.8 mol/striatum) in the rats striatum. Furthermore, these data suggest that Na+,K+-ATPase and GABAA receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1. / O monossialogangliosídeo (GM1) é um componente natural de membrana plasmática que está envolvido no crescimento, reconhecimento e diferenciação celular, além de proteger o SNC da ação dos radicais livres. No presente estudo investigou-se o efeito do GM1 sobre a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD) e glutationa peroxidase (GPx), assim como na quimiluminescência e capacidade antioxidante total (TRAP) em córtex cerebral de ratos machos adultos ex vivo e in vitro. A administração sistêmica de GM1 (50 mg/kg, i.p.; duas doses: 24 horas e 30 minutos antes do sacrifício) reduziu a quimiluminescência e aumentou significativamente a atividade da CAT ex vivo. A adição de GM1 (103-104 nM) ao meio de incubação diminuiu a atividade da CAT in vitro. Estes resultados sugerem que o efeito neuroprotetor do GM1 não é devido à ação antioxidante intrínseca deste glicoesfingolipídeo, mas devido ao aumento secundário das defesas antioxidantes e/ou uma redução da geração de radicais livres. Além disso, avaliamos se o GM1 tinha efeito neuroprotetor em um modelo experimental da acidemia glutárica, um erro inato do metabolismo caracterizado pelo acúmulo tecidual de ácido glutárico (GA) e alterações neurológicas, como degeneração estriatal e convulsões. A administração de GM1 preveniu as convulsões, o aumento da produção dos marcadores do dano oxidativo (carbonilação protéica total e substâncias reativas do ácido tiobarbitúrico - TBARS) e a inibição da atividade da Na+,K+-ATPase induzidas pelo GA (4 mol/2 µl) em estriado de ratos. Além disso, os episódios convulsivos induzidos por GA apresentaram uma correlação significativa com a inibição da atividade da Na+,K+-ATPase no estriado injetado, mas não com os níveis dos marcadores do estresse oxidativo. A adição de GM1 (50 200  ao meio de incubação preveniu a inibição da Na+,K+-ATPase, mas não reduziu o dano oxidativo induzido por GA (6 mM) in vitro. A administração intraestriatal de muscimol (46 pmol/0,5 l), um agonista de receptor GABAA, mas não dos antagonistas de receptores glutamatérgicos, MK-801 (3 nmol/0,5 l) e DNQX (8 nmol/0,5 l), preveniu as convulsões e a inibição da atividade da Na+,K+-ATPase induzidas por GA. A proteção do GM1 e muscimol contra as convulsões induzidas por GA apresentou uma correlação significativa com a manutenção da atividade da Na+,K+-ATPase no estriado injetado com GA. Desde que o GM1 e o muscimol preveniram os efeitos neurotóxicos induzidos pelo GA, investigou-se a ação do GM1 após a administração intraestriatal de pentilenotetrazol (PTZ), um antagonista de receptores GABAA. O tratamento com GM1 preveniu as convulsões, o dano oxidativo e a inibição da atividade da Na+,K+-ATPase induzidas por PTZ (1,8 µmol/2 µl). Esses dados sugerem que a atividade da Na+,K+-ATPase e mecanismos mediados pela ativação de receptores GABAérgicos podem ser de grande importância para a atividade convulsiva induzida por GA, bem como nos mecanismos de neuroproteção induzidos pelo GM1.

This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...

A hipóxia-isquemia (HI) é a principal causa de mortalidade no período perinatal e, nos sobreviventes, a incidência de comorbidades neurológicas é elevada. O encéfalo imaturo, altamente susceptível ao insulto hipóxico-isquêmico, é bastante sensível a estímulos ambientais tais como o enriquecimento ambiental (EA). Os objetivos deste estudo foram: 1) investigar o desempenho comportamental em um novo teste de memória e aprendizagem, o Ox-maze; 2) analisar a atividade das enzimas Na+,K+-ATPase, catalase (CAT) e glutationaperoxidase (GPx) no hipocampo; 3) caracterizar os neurônios piramidais da região CA1 hipocampal quanto à arborização dendrítica; 4) analisar alterações astrocíticas e sinápticas pela avaliação da imunoreatividade das proteínas GFAP e sinaptofisina usando a técnica de imunofluorescência e, 5) quantificar a densidade celular por meio de cortes semifinos da região CA1 do hipocampo de animais hipóxico-isquêmicos expostos a um ambiente enriquecido. Ratos com sete dias de idade foram divididos em quatro grupos e submetidos ou não ao procedimento cirúrgico de acordo com o grupo experimental ao qual pertenciam: controle mantido em ambiente padrão (CTAP), controle em ambiente enriquecido (CTAE), HI em ambiente padrão (HIAP) e HI em ambiente enriquecido (HIAE). Passado o período de EA (1h/dia, 6 dias/semana, 9 semanas iniciando após o desmame), os parâmetros mencionados foram avaliados nos animais. Os dados indicaram que a HI causou um prejuízo na memória e no aprendizado no teste do “OX-maze”, o qual foi revertido pelo efeito do ambiente enriquecido. A HI causou diminuição da atividade enzimática da Na+,K+-ATPase no hipocampo contralateral, assim como uma redução na imunorreatividade à sinaptofisina e nadensidade neuronal, sendo que o EA foi efetivo na recuperação da atividade da enzima Na+,K+-ATPase e dos níveis de sinaptofisina no hipocampo contralateral à lesão. As atividades de CAT e GPX não foram alteradas pela HI em nenhum dos grupos avaliados, mesmo resultado encontrado nas análises de GFAP e de padrão de arborização dendrítica. Por fim, neste estudo foi observado o importante efeito lesivo causado pela HI neonatal e o papel do EA como estratégia neuroprotetora na recuperação funcional, na atividade da Na+,K+-ATPase e na expressão de sinaptofisina. Este estudo traz avanços em busca dos mecanismos pelos quais a melhora funcional ocorre em animais HI expostos ao EA, mas pode-se verificar que não fica totalmente esclarecido como esta estratégia atua. Outros estudos são necessários para a identificação de possíveis mecanismos que atuem como mediadores da resposta funcional do EA após um evento isquêmico. / Hypoxia-ischemia (HI) is the main mortality cause in perinatal period and, in survivors, the incidence of neurological disabilities is elevated. The immature brain, highly susceptible to hypoxic-ischemic insult, is sensible to environmental stimuli, as environmental enrichment (EE). The aims of this study were to investigate: 1) behavioral performance in a new memory and learning task, the oxmaze task; 2) evaluate Na+,K+-ATPase, catalase (CAT) and glutathione peroxidase (GPx) activities in the hippocampus; 3) characterizes dendritic arbor in pyramidal neurons from CA1 region from hippocampus; 4) analyze alterations in hippocampal synaptophysin and GFAP immmunoreactivity and, 5) analyze neuronal density alterations in hippocampus of hypoxic-ischemic rats exposed to enriched environment. Seven-day-old rats were divided into four groups: controlmaintained in standard environment (CTSE), control submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Past the end of EE period (1 hour/day, 6 days/week, 9 weeks), mentioned parameters were evaluated in animals. Present results indicate learning and memory in the “OXmaze” task were impaired in HI rats and this effect was recovered after EE. On the contralateral hemisphere, HI caused a decrease in Na+,K+-ATPase activity that was recovered by EE. Results also indicate that HI damage decreases hippocampal synaptophysin immunoreactivity and neuronal density, moreover EE was effective in recovering synaptophysin levels on contralateral to the lesion hippocampus. The activities of GPx and CAT were not changed by HI in any group evaluated, some result founded on GFAP immunoreactivity and dendritic arborization characterization analysis. In conclusion, the important effect of HI lesion and the role of EE like neuroprotective strategy on functional impairment and on Na+,K+-ATPase activity and synaptophysin immunoreactivity was proven. Although this study have important advances in search of mechanisms by which the functional enhancement occurs in the animals submitted to HI and exposed to EE, it can be seen that it is not completely clear how this approach works. Further studies are needed to identify possible mechanisms that act as mediators of EE functional response after an ischemic event.

Estudos prévios demonstram que intervenções precoces levam a alterações comportamentais e neuroendócrinas na vida adulta. Nossos achados anteriores demonstram que animais manipulados no período neonatal consomem mais alimento palatável na vida adulta e apresentam um menor aumento do depósito de gordura abdominal, após exposição crônica a dieta palatável (chocolate) em relação aos animais intactos. Neste trabalho de tese, nosso objetivo foi avaliar os efeitos da manipulação neonatal sobre a preferência alimentar bem como sobre a regulação metabólica de ratas adultas. Para isso, investigamos parâmetros metabólicos e neuroquímicos em resposta à exposição crônica a uma dieta hipercalórica e palatável, assim como a um período de abstinência desse tipo de alimento em ratas fêmeas adultas expostas ou não à manipulação neonatal (10 min/dia, 10 primeiros dias de vida). A manipulação neonatal induziu maior ingestão de alimento palatável após um curto período de privação. Entretanto, o consumo durante a exposição crônica a essa dieta não diferiu entre os grupos experimentais. Também observamos que ratas fêmeas manipuladas, quando cronicamente expostas à dieta palatável na vida adulta, têm menor aumento da gordura abdominal e esse efeito persiste após a privação da dieta. Ratas não-manipuladas apresentaram níveis mais elevados de colinesterases no soro após exposição crônica a dieta palatável, entretanto, sem alterações na atividade de colinesterases no córtex cerebral. Foi identificado, após exposição crônica a dieta palatável, uma redução na atividade da enzima Na+,K+-ATPse no hipocampo e na amígdala e um aumento nos níveis plasmáticos de S100B em ratas não-manipuladas no período neonatal. Após as primeiras 24h de privação do alimento palatável, ratas fêmeas não-manipuladas demonstraram maior frequência de sinais de abstinência (tremores de cabeça) em comparação com ratas manipuladas no período neonatal. Assim, esses achados sugerem que a manipulação neonatal determina alterações persistentes no comportamento alimentar e previne algumas alterações periféricas e centrais induzidas pela exposição crônica a uma dieta hiperpalatável, modulando a resposta metabólica de modo a reduzir a vulnerabilidade de dano metabólico e neural. / Previous studies have demonstrated that an intervention early in life leads to behavior and neuroendocrine alterations in adulthood. According to our previous findings neonatallyhandled animals have an increased consumption of palatable food, as well as a lower increase in abdominal fat accumulation after being chronically exposed to a highly palatable diet (chocolate) as compared with intact rats. In the present study, our aim was to evaluate the effects of neonatal handling on food preference and metabolic regulation in adult female rats. Therefore, we investigated metabolic and neurochemical parameters in response to a chronic exposure to a highly palatable diet, and to its withdrawal in adult female rats exposed or not to neonatal handling procedure (10 min/day, 10 first days of life). We observed an effect of neonatal handling inducing an increased palatable food intake after one week of chocolate withdrawal. However, chocolate consumption during long-term exposure to this type of diet did not differ between experimental groups. After a 30-days-period of chocolate exposure, non-handled female rats exhibited an increased abdominal fat deposition in comparison to neonatally-handled rats, and this effect persisted even after chocolate withdrawal. Nonhandled rats had increased serum cholinesterase levels after chronic exposure to palatable diet, without alterations in cerebral cortex cholinesterase activity. We also observed that chocolate consumption lead to a reduced Na+,K+-ATPse activity in hippocampus and amygdala, as well as an increased plasma S100B levels in non-handled females rats. After the first 24h of chocolate withdrawal, non-handled female rats exhibited an increased frequency of head shakes, during the Open Field task, in comparison to handled rats. Therefore, these findings suggest that neonatal handling leads to persistent alterations in feeding behavior, and also prevents some peripheral and central alterations induced by chronic exposure to a highly palatable diet; modulating the metabolic response in order to reduce the vulnerability to metabolic and neuronal damage in adulthood.

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.

A hipóxia-isquemia (HI) é a principal causa de mortalidade no período perinatal e, nos sobreviventes, a incidência de comorbidades neurológicas é elevada. O encéfalo imaturo, altamente susceptível ao insulto hipóxico-isquêmico, é bastante sensível a estímulos ambientais tais como o enriquecimento ambiental (EA). Os objetivos deste estudo foram: 1) investigar o desempenho comportamental em um novo teste de memória e aprendizagem, o Ox-maze; 2) analisar a atividade das enzimas Na+,K+-ATPase, catalase (CAT) e glutationaperoxidase (GPx) no hipocampo; 3) caracterizar os neurônios piramidais da região CA1 hipocampal quanto à arborização dendrítica; 4) analisar alterações astrocíticas e sinápticas pela avaliação da imunoreatividade das proteínas GFAP e sinaptofisina usando a técnica de imunofluorescência e, 5) quantificar a densidade celular por meio de cortes semifinos da região CA1 do hipocampo de animais hipóxico-isquêmicos expostos a um ambiente enriquecido. Ratos com sete dias de idade foram divididos em quatro grupos e submetidos ou não ao procedimento cirúrgico de acordo com o grupo experimental ao qual pertenciam: controle mantido em ambiente padrão (CTAP), controle em ambiente enriquecido (CTAE), HI em ambiente padrão (HIAP) e HI em ambiente enriquecido (HIAE). Passado o período de EA (1h/dia, 6 dias/semana, 9 semanas iniciando após o desmame), os parâmetros mencionados foram avaliados nos animais. Os dados indicaram que a HI causou um prejuízo na memória e no aprendizado no teste do “OX-maze”, o qual foi revertido pelo efeito do ambiente enriquecido. A HI causou diminuição da atividade enzimática da Na+,K+-ATPase no hipocampo contralateral, assim como uma redução na imunorreatividade à sinaptofisina e nadensidade neuronal, sendo que o EA foi efetivo na recuperação da atividade da enzima Na+,K+-ATPase e dos níveis de sinaptofisina no hipocampo contralateral à lesão. As atividades de CAT e GPX não foram alteradas pela HI em nenhum dos grupos avaliados, mesmo resultado encontrado nas análises de GFAP e de padrão de arborização dendrítica. Por fim, neste estudo foi observado o importante efeito lesivo causado pela HI neonatal e o papel do EA como estratégia neuroprotetora na recuperação funcional, na atividade da Na+,K+-ATPase e na expressão de sinaptofisina. Este estudo traz avanços em busca dos mecanismos pelos quais a melhora funcional ocorre em animais HI expostos ao EA, mas pode-se verificar que não fica totalmente esclarecido como esta estratégia atua. Outros estudos são necessários para a identificação de possíveis mecanismos que atuem como mediadores da resposta funcional do EA após um evento isquêmico. / Hypoxia-ischemia (HI) is the main mortality cause in perinatal period and, in survivors, the incidence of neurological disabilities is elevated. The immature brain, highly susceptible to hypoxic-ischemic insult, is sensible to environmental stimuli, as environmental enrichment (EE). The aims of this study were to investigate: 1) behavioral performance in a new memory and learning task, the oxmaze task; 2) evaluate Na+,K+-ATPase, catalase (CAT) and glutathione peroxidase (GPx) activities in the hippocampus; 3) characterizes dendritic arbor in pyramidal neurons from CA1 region from hippocampus; 4) analyze alterations in hippocampal synaptophysin and GFAP immmunoreactivity and, 5) analyze neuronal density alterations in hippocampus of hypoxic-ischemic rats exposed to enriched environment. Seven-day-old rats were divided into four groups: controlmaintained in standard environment (CTSE), control submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Past the end of EE period (1 hour/day, 6 days/week, 9 weeks), mentioned parameters were evaluated in animals. Present results indicate learning and memory in the “OXmaze” task were impaired in HI rats and this effect was recovered after EE. On the contralateral hemisphere, HI caused a decrease in Na+,K+-ATPase activity that was recovered by EE. Results also indicate that HI damage decreases hippocampal synaptophysin immunoreactivity and neuronal density, moreover EE was effective in recovering synaptophysin levels on contralateral to the lesion hippocampus. The activities of GPx and CAT were not changed by HI in any group evaluated, some result founded on GFAP immunoreactivity and dendritic arborization characterization analysis. In conclusion, the important effect of HI lesion and the role of EE like neuroprotective strategy on functional impairment and on Na+,K+-ATPase activity and synaptophysin immunoreactivity was proven. Although this study have important advances in search of mechanisms by which the functional enhancement occurs in the animals submitted to HI and exposed to EE, it can be seen that it is not completely clear how this approach works. Further studies are needed to identify possible mechanisms that act as mediators of EE functional response after an ischemic event.