Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

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Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-15Bitstream added on 2014-06-13T20:06:46Z : No. of bitstreams: 1 diego_las_dr_botfm.pdf: 928445 bytes, checksum: b041c5d0355031145492120fceebec02 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A glibenclamida, sulfoniluréia muito utilizada clinicamente no controle do diabetes mellitus, possui propriedade bloqueadora dos canais de potássio dependentes de adenosina trifosfatona (k+ ATP), opondo-se à possível propriedade pré-condicionante do sevoflurano. Hipovolemia conseqüente à hemorragia suscita reações compensadoras de proteção orgânica. Entretanto, mecanismos protetores como a ativação do sistema renina-angiotensina ensejam agressão renal por vasoconstrição com interferência na função depuradora. O objetivo do presente estudo foi avaliar os efeitos da glibenclamida na função e histologia renais de ratos sob anestesia com sevoflurano em cenário de choque hemorrágico. Neste intuito, estudou-se uma amostra de 20 ratos Wistar. O delineamento consistiu em estudar os animais quanto à pressão arterial, à temperatura retal e ao hematócrito e quantificar atributos da função renal, tais como: ritmo de filtração glomerular (RGF), estudar os animais quanto ao peso, pressão arterial média, temperatura retal e hematócrito e fluxo plasmático renal efetivo (FPR efetivo), fluxo sangüíneo renal e resistência vascular renal (RVR), além de se analisarem as alterações histológicas após a imposição da condição experimental. A amostra foi aleatoriamente dividida em 2 grupos de 10 animais cada (G1 e G2), entretanto apenas um destes (G2) foi submetido a tratamento com glibenclamida venosa (1 mcg.g-1), 60 min antes do início de sangria. Os animais de ambos os grupos foram anestesiados com sevoflurano a 4% sob campânula apropriada e mantidos em respiração espontânea com ar enriquecido com oxigênio, além de sofrerem sangria de 30% da volemia (esta calculada como 6% do peso corporal)... / Glibenclamide, a sulfonylurea widely used clinically for controlling diabetes mellitus, blocks adenosine triphosphate-dependent potassium (K+ ATP) channels, thus opposing the possible preconditioning property of sevoflurane. Hypovolemia from hemorrhage evokes protective compensatory reactions. However, protective mechanisms such as activation of the renin-angiotensin system result in renal impairment by vasoconstriction interfering in the clearance function. This study was designed to evaluate the effects of glibenclamide on renal function and histology in rats in a state of hemorrhagic shock under sevoflurane anesthesia. A sample of 20 Wistar rats was studied for that purpose. The design consisted in studying mean arterial pressure, rectal temperature and hematocrit and quantifying glomerular filtration rate (GFR), effective renal plasma flow (ERPF), renal blood flow (RBF) and renal vascular resistance (RVR), and analyzing histological alterations after the experimental conditions were attained. The sample was randomized into 2 groups of 10 animals each (G1 and G2), only one of which (G2) was treated with intravenous glibenclamide (1 mcg.g-1), 60 min before bleeding was begun. Both groups were anesthetized with sevoflurane at 4% under an appropriate bell jar and kept on spontaneous respiration with oxygen-enriched air, while being bled of 30% of volemia (calculated as 6% of body weight) in 3 stages with 10-min intervals. Evaluation of renal function used estimated renal clearances of paraaminohippurate (ERPF) and sodium iothalamate (GFR), while renal histology was evaluated by investigating the degree of vascular and tubular dilatation, vascular congestion, tubular vacuolization, necrosis and signs of tubular regeneration... (Complete abstract click electronic access below)

Orientador: Yara Marcondes Machado Castiglia / Banca: Yara Marcondes Machado Castiglia / Banca: Pedro Thadeu Galvão Vianna / Banca: Luis Antonio Vane / Banca: José Luiz Gomes do Amaral / Banca: Luís Vicente Garcia / Resumo: A glibenclamida, sulfoniluréia muito utilizada clinicamente no controle do diabetes mellitus, possui propriedade bloqueadora dos canais de potássio dependentes de adenosina trifosfatona (k+ ATP), opondo-se à possível propriedade pré-condicionante do sevoflurano. Hipovolemia conseqüente à hemorragia suscita reações compensadoras de proteção orgânica. Entretanto, mecanismos protetores como a ativação do sistema renina-angiotensina ensejam agressão renal por vasoconstrição com interferência na função depuradora. O objetivo do presente estudo foi avaliar os efeitos da glibenclamida na função e histologia renais de ratos sob anestesia com sevoflurano em cenário de choque hemorrágico. Neste intuito, estudou-se uma amostra de 20 ratos Wistar. O delineamento consistiu em estudar os animais quanto à pressão arterial, à temperatura retal e ao hematócrito e quantificar atributos da função renal, tais como: ritmo de filtração glomerular (RGF), estudar os animais quanto ao peso, pressão arterial média, temperatura retal e hematócrito e fluxo plasmático renal efetivo (FPR efetivo), fluxo sangüíneo renal e resistência vascular renal (RVR), além de se analisarem as alterações histológicas após a imposição da condição experimental. A amostra foi aleatoriamente dividida em 2 grupos de 10 animais cada (G1 e G2), entretanto apenas um destes (G2) foi submetido a tratamento com glibenclamida venosa (1 mcg.g-1), 60 min antes do início de sangria. Os animais de ambos os grupos foram anestesiados com sevoflurano a 4% sob campânula apropriada e mantidos em respiração espontânea com ar enriquecido com oxigênio, além de sofrerem sangria de 30% da volemia (esta calculada como 6% do peso corporal)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glibenclamide, a sulfonylurea widely used clinically for controlling diabetes mellitus, blocks adenosine triphosphate-dependent potassium (K+ ATP) channels, thus opposing the possible preconditioning property of sevoflurane. Hypovolemia from hemorrhage evokes protective compensatory reactions. However, protective mechanisms such as activation of the renin-angiotensin system result in renal impairment by vasoconstriction interfering in the clearance function. This study was designed to evaluate the effects of glibenclamide on renal function and histology in rats in a state of hemorrhagic shock under sevoflurane anesthesia. A sample of 20 Wistar rats was studied for that purpose. The design consisted in studying mean arterial pressure, rectal temperature and hematocrit and quantifying glomerular filtration rate (GFR), effective renal plasma flow (ERPF), renal blood flow (RBF) and renal vascular resistance (RVR), and analyzing histological alterations after the experimental conditions were attained. The sample was randomized into 2 groups of 10 animals each (G1 and G2), only one of which (G2) was treated with intravenous glibenclamide (1 mcg.g-1), 60 min before bleeding was begun. Both groups were anesthetized with sevoflurane at 4% under an appropriate bell jar and kept on spontaneous respiration with oxygen-enriched air, while being bled of 30% of volemia (calculated as 6% of body weight) in 3 stages with 10-min intervals. Evaluation of renal function used estimated renal clearances of paraaminohippurate (ERPF) and sodium iothalamate (GFR), while renal histology was evaluated by investigating the degree of vascular and tubular dilatation, vascular congestion, tubular vacuolization, necrosis and signs of tubular regeneration... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:29:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-19Bitstream added on 2014-06-13T18:59:01Z : No. of bitstreams: 1 marques_co_me_botfm.pdf: 378901 bytes, checksum: 2a06b8e8fb2e48ad5f6c9ce7c12f4c38 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Choque hemorrágico reduz o fluxo sanguíneo e a oxigenação teciduais, bem como a remoção de produtos de degradação. A hipóxia tecidual provoca alteração na síntese e liberação de citocinas pró-inflamatórias. A glibenclamida, antagonista dos canais KATP, em rins de ratos anestesiados com sevoflurano e que sofreram hemorragia, preservou mais a histologia e a função renais. O objetivo deste estudo foi verificar se houve alterações da concentração sérica e da expressão renal das citocinas IL-1 e TNF- em ratos que receberam sevoflurano e glibenclamida e que sofreram hemorragia, sem reposição adequada da volemia. Dois grupos de ratos Wistar (n=10) anestesiados com sevoflurano: G1, controle, e G2, com glibenclamida, 1μg/g iv, submetidos à hemorragia de 30% da volemia (10% a cada 10 min), com reposição por Ringer lactato, 5mL/kg/h. Estudaram-se as concentrações séricas de IL-1 e TNF- : na primeira hemorragia (M1) e 50 min após (M2). Em M2, estudou-se a expressão renal dessas citocinas. Em G1, TNF- sérico (normal de 143pg/ml): M1=178,6pg/mL ± 33,5 e M2=509,2pg/mL ± 118,8 e IL-1 sérica (normal de 158pg/mL): M1=148,8pg/mL ± 31,3 e M2=322,6pg/mL ± 115,4. Em G2, TNF- sérico: M1=486,2pg/mL ± 83,6 e M2=261,8pg/mL ± 79,5 e IL-1 sérica: M1=347,0pg/mL ± 72,0 e M2=327,3pg/mL ± 90,9. Houve expressão de TNF- e IL-1 nas células tubulares e glomerulares renais, mais marcantes em G2. A hemorragia e glibenclamida aumentaram a concentração sérica e a imunomarcação renal das citocinas IL-1 e TNF- , mas G2 enfrentou a hipotensão conseqüente à hemorragia com vasodilatação (provável ação do TNF- ) e melhor perfusão, resultando em alguma proteção / Hemorrhagic shock reduces blood flow and tissue oxygenation, as well as the removal of degradation products. Tissue hypoxia causes changes in the synthesis and release of proinflammatory cytokines. Treatment with the KATP channel antagonist glibenclamide in sevoflurane-anesthetized rats that suffered from hemorrhaging preserved more renal function and histology. The objective of this study was to verify if there were changes in the serum concentration and renal expression of the IL-1 and TNF- cytokines in rats that received sevoflurane and glibenclamide and had hemorrhaging with inadequate volemia replacement. Two groups of sevofluraneanesthetized Wistar rats (n=10): G1 (control) and G2 (with glibenclamide, 1 μg/g i.v.) were subjected to 30% blood volume hemorrhaging (10% every 10 min), with replacement using Ringer’s lactate, 5 ml/kg/h. The IL-1 and TNF- serum concentrations were studied in the first hemorrhage (M1) and then 50 min later (M2). At M2, the renal expression of these cytokines was also studied. In G1, serum TNF- (normal range around 143 pg/mL) was M1=178.6 ± 33.5 pg/mL and M2=509.2 ± 118.8 pg/mL, while serum IL-1 (normal range around 158 pg/mL) was M1=148.8 ± 31.3 pg/mL and M2=322.6 ±115.4 pg/mL. In G2, serum TNF- was M1=486.2 ± 83.6 pg/mL and M2=261.8 ± 79.5 pg/mL, and serum IL-1 was M1=347.0 ± 72.0 pg/mL and M2=327.3 ±90.9 pg/mL. The expression of TNF- and IL-1 in the renal glomerular and tubular cells was more abundant in the G2 group. Hemorrhage and glibenclamide increased the serum concentration and renal immunoreactions of the TNF- and IL-1 cytokines, but the G2 group experienced hypotension resulting from the hemorrhage with vasodilatation, probably due to TNF- , and better perfusion, resulting in some protection

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at -70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (K(Ca)) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K(Ca) activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V(m)) and 145 ± 13 pS at negative V(m). Glibenclamide (50 μM), Ba2+ (1 mM) and quinine (100 μM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 μM. The identity of this K+ channel with an ATP-sensitive K+ channel (K(ATP)) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 μM). We conclude that plasma membranes of HTB-9 cells contain the K(Ca) and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked K(ATP).

Le canal sodique épithélial, ENaC, est une protéine hétéromérique constituée de trois sous-unités: [alpha], [bêta] et [gamma]. ENaC est exprimé au niveau de la membrane apicale des cellules épithéliales du néphron, du colon distal et des voies aériennes. Au niveau du néphron, l'activité et l'expression de ENaC sont finement régulées par deux hormones: l'aldostérone et la vasopressine, ce qui fait de ENaC un élément clé du maintient de la pression artérielle. ENaC joue également un rôle dans la régulation de la pression artérielle dans le colon distal. Il existe plusieurs mutations du canal directement liées à des troubles graves de la pression artérielle: le syndrome de Liddle et le pseudoaldostéronisme de type I (PHA I). La première pathologie est un gain de fonction du canal provoquant une hypertension précoce et sévère alors que le PHA I est une perte de fonction caractérisée par une diminution de la réabsorption sodium et une hypotension. ENaC est également une cible thérapeutique de choix dans les hypertensions de type 1. Il a récemment été démontré que la sous-unité [alpha] ENaC était impliquée dans l'augmentation de l'activité du canal de cobaye mais pas de rat par deux ligands extra-cellulaires: le cpt-cAMP un analogue perméant de l'AMPc, et le glibenclamide, un inhibiteur des canaux K[indice inférieur ATP]. Notre objectif est de déterminer les sites d'interaction des ces ligands avec ENaC ce qui permettra de mieux comprendre la relation structure/fonction de la boucle extra-cellulaire. Des techniques de biologie moléculaire nous ont permis de construire des sous-unités [alpha] chimériques contenant des séquences de rat et de cobaye. Ces chimères ont été coexprimées dans l'ovocyte de xénope avec les sous-unités [bêta] rat et [gamma] rat et les courants macroscopiques ont été enregistré en voltage clamp à deux électrodes. Nous avons ainsi déterminé que les régions centrales et terminales de la boucle extra-cellulaire de la sous-unité [alpha]gp conféraient une sensibilité partielle au cpt-cAMP. La réalisation de mutants nous a permis d'identifier cinq acides aminés dans la région terminale de la boucle dont une isoleucine en position 510 indispensable à l'activation du canal par le cpt-cAMP. Nous avons montré en patch clamp en configuration"outside out" que cette augmentation du courant macroscopique résultait d'une augmentation de la NPo du canal et non de sa conductance unitaire. Concernant l'interaction entre le glibenclamide et la sous-unité [alpha], nous avons employé la même approche que pour le cpt-cAMP. Nos résultats préliminaires indiquent que la totalité de la boucle extra-cellulaire est impliquée dans l'interaction avec le glibenclamide. Comme pour le cpt-cAMP, la région terminale de la boucle extra-cellulaire confère la plus grande sensibilité au ligand. Cependant, nous n'avons pas identifié tous les acides aminés interagissant avec le glibenclamide. Nous soupçonnons l'isoleucine 510 de jouer un rôle dans cette interaction.

Background: Modulation of ischemia-dependent pathways alters electrophysiological evolution of ventricular fibrillation(VF). Hypothesis: 1)There is regional disease-related expression of KATP-channels in human cardiomyopathic hearts. 2)KATP-channel blockade promotes spontaneous VF termination by attenuating spatiotemporal dispersion of refractoriness(ΔERP). Methods and Results: Electric mapping of control(n=6) and treatment(n=9) (10 μmol/L glibenclamide) isolated human cardiomyopathic hearts was performed. Spontaneous defibrillation and KATP-subunit gene expression were studied. Spontaneous VF termination occurred in 1/6 control and 7/8 treated hearts (P=0.026). After 180 seconds of ischemia, LV transmural dispersion in VF cycle length was observed(p=0.001), which was attenuated by glibenclamide. There was greater gene expression of all KATP-subunit on the endocardium compared with the epicardium(P<0.02). In ischemic rat heart model, ΔERP was verified with pacing protocols (36±5ms vs 4.9±4ms, p=0.019). Conclusions: KATP channel subunit gene expression is heterogeneously altered in the cardiomyopathic human heart. Blockade of KATP channels promotes spontaneous defibrillation by attenuating ischemia-dependent ΔERP during VF.

Background: Modulation of ischemia-dependent pathways alters electrophysiological evolution of ventricular fibrillation(VF). Hypothesis: 1)There is regional disease-related expression of KATP-channels in human cardiomyopathic hearts. 2)KATP-channel blockade promotes spontaneous VF termination by attenuating spatiotemporal dispersion of refractoriness(ΔERP). Methods and Results: Electric mapping of control(n=6) and treatment(n=9) (10 μmol/L glibenclamide) isolated human cardiomyopathic hearts was performed. Spontaneous defibrillation and KATP-subunit gene expression were studied. Spontaneous VF termination occurred in 1/6 control and 7/8 treated hearts (P=0.026). After 180 seconds of ischemia, LV transmural dispersion in VF cycle length was observed(p=0.001), which was attenuated by glibenclamide. There was greater gene expression of all KATP-subunit on the endocardium compared with the epicardium(P<0.02). In ischemic rat heart model, ΔERP was verified with pacing protocols (36±5ms vs 4.9±4ms, p=0.019). Conclusions: KATP channel subunit gene expression is heterogeneously altered in the cardiomyopathic human heart. Blockade of KATP channels promotes spontaneous defibrillation by attenuating ischemia-dependent ΔERP during VF.