Current therapeutic options for ischemic stroke are limited to tissue plasminogen activator and mechanical clot removal therapies. Diazoxide (DZ) is a mitochondrial ATP-sensitive potassium channel opener and is protective in models of brain ischemia, but the signaling pathways involved are unknown. The mammalian target of rapamycin (mTOR) is a master regulator of protein synthesis and is involved in protection against cerebral ischemia. The neuronal nitric oxide synthase (nNOS) pathway has also been shown to provide protection from ischemic insults. Additionally, mitochondrial respiratory status has not been investigated. I examined the role of the mTOR pathway, the nNOS pathway, and mitochondrial respiration in delayed DZ-induced preconditioning of neurons. I cultured rat primary cortical neurons and simulated ischemic stroke using oxygen-glucose deprivation (OGD) for 3 h followed by re-oxygenation. Viability, mitochondrial membrane potential, reactive oxygen species (ROS) measurements, and western blots were performed. The mTOR pathway was inhibited by rapamycin, Torin-1, and S6K targeted silencing RNA. The NOS pathway was inhibited by L-NAME. NO-donors SNP and DEANONOate (DEANO) were applied to rescue the effects of L-NAME. Mitochondrial oxygen consumption rate (OCR) was measured in intact neurons by serial injections of oligomycin, FCCP, and antimycin/rotenone. OGD decreased viability by 50 percent, depolarized mitochondria, and reduced mitochondrial respiration whereas DZ improved viability to 75 percent and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Diazoxide also increased phosphorylation of protein kinase B, mTOR, and S6K. Rapamycin, Torin-1, and S6K targeted siRNA abolished the protective effects of DZ. Co-application of L-NAME with DZ prevented preconditioning whereas adding SNP or DEANO along with L-NAME and DZ restored protection. Diazoxide increased phosphorylated nNOS. Interestingly, co-application of LNAME with DZ blocked the phosphorylation of nNOS as well as S6K. The ratio of phosphorylated/total Akt and mTOR were not significantly altered with L-NAME co-application. Diazoxide altered OCR 24 and 48 h after the ischemic period. Diazoxide had no acute effect on OCR but increased ECAR significantly. Activation of the mTOR and nNOS pathways is critical for DZ preconditioning in neurons. Furthermore, OCR is modified by the DZ-induced preconditioning of neurons. / acase@tulane.edu

nÃo hà / A pentoxifilina (PTX) à um inibidor nÃo-seletivo da fosfodiesterase, com aÃÃes antiinflamatÃrias vasculares e reolÃgicas que podem neutralizar algumas das mudanÃas no diabetes mellitus (DM) que contribuem para amenizar os seus efeitos secundÃrios como a neuropatia, a retinopatia e a nefropatia. Tendo em vista as propriedades antiinflamatÃrias da pentoxifilina e o envolvimento da inflamaÃÃo com o DM, buscou-se investigar seus possÃveis efeitos hipoglicemiante e hipolipemiante no modelo de DM induzido por aloxano em ratos. A pentoxifilina (PTX) em estudos pilotos apresentou efeito hipoglicemiante e reduziu os nÃveis de triglicerÃdeos em animais com diabetes induzidos por aloxano, nas doses 5, 25, 50 e 100 mg/Kg. A administraÃÃo oral da associaÃÃo de pentoxifilina (PTX) com glibenclamida (GLI), PTX5 + GLI2, causou reduÃÃes significativas nos nÃveis plasmÃticos de glicose e triglicerÃdeos em curto e longo prazo, evidenciando que o mecanismo de aÃÃo da PTX pode ser explicado via canais de K+ATP-dependentes. A administraÃÃo oral da associaÃÃo de Pentoxifilina (PTX) com Metformina (MET), PTX5 + MET5, ocasionou uma reduÃÃo da hiperglicemia apenas a longo prazo, sugerindo nÃo compartilharem o mesmo mecanismo. A PTX nÃo bloqueou a hiperglicemia induzida pelo Diazoxido (DZD), um antagonista da GLI, que inibe a secreÃÃo de insulina prolongando o tempo de abertura dos canais de K+ATP-dependentes, sugerindo que outros fatores, alÃm do bloqueio de canais de K+-ATP dependentes, podem estar envolvidos. A reduÃÃo nos valores de hemoglobina glicada (A1C) e de frutosamina mostraram que o tratamento com PTX50 e com a associaÃÃo PTX5+GLI2 melhorou o controle glicÃmico dos animais em estudo, indicando que esta droga pode inibir o desenvolvimento de lesÃes micro e macrovasculares advindas do DM. A PTX mostrou um marcante efeito antiinflamatÃrio, melhorando o estado geral dos ratos em experimentaÃÃo. Reduziu, de forma significativa, o edema de pata nas doses de 50 e 100 mg/Kg, todavia foi visto que o perfil inflamatÃrio no rato diabÃtico tem um padrÃo diferenciado do rato normal, evidenciando uma amplificaÃÃo do processo inflamatÃrio no rato diabÃtico quando comparado ao rato normal. Foi visto ainda que os nÃveis de TNF-∝ e IL-6 aumentaram de modo significante apÃs a induÃÃo do edema de pata nos ratos diabÃticos, entretanto nos ratos tratados com PTX os nÃveis teciduais destas citocinas mostraram-se significativamente mais baixos, o que fala a favor de uma evidente aÃÃo antiinflamatÃria da PTX. A PTX mostrou tambÃm um importante efeito antioxidante reduzindo, de forma significativa, as liberaÃÃes de nitrito tecidual e sÃrica, atuando favoravelmente na reduÃÃo de radicais livres. O tratamento prolongado com PTX foi eficaz em manter o padrÃo normal do pÃncreas, do fÃgado e dos rins nos grupos diabÃticos tratados com PTX50 e com PTX5+GLI2, indicando uma aÃÃo protetora da PTX contra a citotoxicidade induzida pelo aloxano. Os efeitos hipoglicemiante e hipotrigliceridÃmico da PTX, aqui demonstrados, podem està correlacionado com sua aÃÃo sobre o estresse oxidativo e sobre a low grade inflammation, o que torna a PTX um importante alvo terapÃutico para o manejo do diabetes mellitus na clÃnica / Pentoxifylline (PTX) is a non-selective inhibitor of phosphodiesterase with anti-inflammatory vascular and rheological properties. The drug can neutralize some of the changes seen in diabetes mellitus (DM), contributing to attenuate diabetes secondary complications as neuropathy, retinopathy and nephropathy. Considering PTX anti-inflammatory properties and the known involvement of inflammation with DM, we investigated its possible hypoglycemic and hypolipidemic effects in the model of alloxan-induced DM in rats. Pentoxifylline pilot studies in reduced plasma levels of glucose and triglycerides in animals with diabetes induced by alloxan at the doses of 5, 25, 50 and 100 mg/kg. Oral administration of the combination of PTX with glibenclamide (GLI), PTX5 + GLI2, caused significant reductions in plasma levels of glucose and triglycerides in the short and long term, indicating that the mechanism of action of PTX can be explained via K+ATP-dependent channels. The oral administration of the combination of pentoxifylline (PTX) with metformin (MET), PTX5+MET5, caused a reduction of only the long term hyperglycemia, suggesting that these two drugs do not share the same mechanism. PTX did not block the hyperglycemia induced by diazoxide (DZD), an antagonist of GLI, which inhibits insulin secretion by prolonging the opening time of the K+ATP-dependent-channel. This result suggests that other factors, in addition to the blockade of the K+ATP dependent channels, may be involved. The reduction in glycosylated hemoglobin (A1C), and fructosamine showed that treatment with the combination PTX5+GLI2 and PTX50, improved glycemia in the study, indicating that this drug can inhibit the development of macrovascular and microvascular injury resulting from DM. The PTX showed a marked anti-inflammatory effect, improving the general condition of rats subjected to acute inflammation models. PTX reduced significantly, the paw edema at doses of 50 and 100 mg / kg, However, the inflammatory profile in diabetic rats have a different pattern of that seen in non-diabetic rat, showing an amplification of the inflammatory process. We showed that the levels of TNF-α and IL-6 were significantly increased after the induction of paw edema in diabetic rats, but in the rats treated with PTX tissue levels of these cytokines were significantly lower, which indicating a clear anti-inflammatory action of PTX. PTX also showed a significant antioxidant effect reducing significantly the release of tissue and serum nitrite, acting favorably in the reduction of free radicals. The prolonged treatment with PTX was effective in maintaining the normal histological pattern of the pancreas, liver and kidneys in diabetic groups treated with PTX50 PTX5 + and GLI2, indicating a protective effect of PTX against alloxan-induced cytotoxicity. The hypoglycemic and hypotriglyceridemic effects of PTX, shown here, may correlate with its effect on oxidative stress and on low grade inflammation, making PTX an important candidate for the management of diabetes mellitus in the clinic

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2006. / Typescript (photocopy).

Diazoxide, a non-diuretic benzothiadiazlne antihypertensive agent, is thought to act directly upon the vascular smooth muscle of the resistance vessels to exert its therapeutic effects in hypertension. Diazoxide may exert its antihypertensive action by antagonizing calcium in vascular smooth muscle. Wohl et al. (1967 and 1968) have suggested such an interaction based on experiments conducted with isolated rabbit aortae. The present experiments were designed to investigate the possible cellular locus of the postulated interaction of diazoxide with calcium using the isolated anterior mesenteric vein of the rabbit as a model of vascular smooth muscle. This vein is spontaneously motile and possesses characteristics similar to those observed for vessels of the microcirculation. Diazoxide at 10ˉ⁴ M inhibited spontaneous motility and its associated membrane electrical activity, and caused hyperpolarization in rabbit anterior mesenteric veins examined with a sucrose gap apparatus. Diazoxide also inhibited spontaneous electrical and contractile activity in guinea-pig taenia coli and in estrogen dominated rabbit uterus. In all these tissues, calcium is believed to play an important role in spontaneous electrical membrane activity. Diazoxide failed to affect contractility, rate of spontaneous contractions, or action potential configurations in isolated rabbit heart, even though the action potential in heart tissues possesses a definite calcium current component. Diazoxide reduced contractions induced in the mesenteric vein by electrical stimulation of the smooth muscle itself or by excitation of the nerve endings within the vein. Various drugs were chosen for their ability to contract the mesenteric vein in different ways. Noradrenaline contracts vascular smooth muscle even when the tissue Is depolarized with ouabain Diazoxide failed to inhibit noradrenaline contractions in the depolarized vein, but showed the characteristics of a competitive inhibitor of noradrenaline in normally polarized veins. Diazoxide was also capable of inhibiting contractions to serotonin and procaine, agents which require membrane polarization to initiate contraction. The inhibitory effect of diazoxide was not observed to be modified in solutions containing high concentrations of calcium. Diazoxide was tested upon the contractile responses to calcium In veins depolarized in K⁺ Ringer solution. Examination of the resultant dose response curves showed that diazoxide inhibited calcium contractions ln a reversible, non surmountable manner. Hydrochlorothiazide had no effect upon calcium induced contractions. Diazoxide antagonizes drug induced contractions only if a polarized membrane is present. Calcium Induced contractions in depolarizing solutions were inhibited in an apparently Insurmountable manner, while drug responses in polarizing solutions were inhibited by diazoxide in a surmountable manner. In addition, action potentials from rabbit heart were unchanged whereas, the apparently calcium spike mediated electrical activity of certain smooth muscles is inhibited. It is concluded that diazoxide affects the membrane of vascular smooth muscle to reduce excitability of the tissue to drugs or electrical stimuli. It is possible that cell membrane bound calcium could be the locus of action of diazoxide and that this agent modifies membrane calcium to cause increased membrane stability. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

No / Although ATP-sensitive potassium (K(ATP)) channel openers, e.g., minoxidil and diazoxide, can induce hair growth, their mechanisms require clarification. Improved drugs are needed clinically. but the absence of a good bioassay hampers research. K(ATP) channels from various tissues contain subtypes of the regulatory sulfonylurea receptor, SUR, and pore-forming, K(+) inward rectifier subunits, Kir6.X, giving differing sensitivities to regulators. Therefore, the in vitro effects of established potassium channel openers and inhibitors (tolbutamide and glibenclamide), plus a novel, selective Kir6.2/SUR1 opener, NNC 55-0118, were assessed on deer hair follicle growth in serum-free median without streptomycin. Minoxidil (0.1-100 microM, p<0.001), NNC 55-0118 (1 mM, p<0.01; 0.1, 10, 100 microM, p<0.001), and diazoxide (10 microM, p<0.01) increased growth. Tolbutamide (1 mM) inhibited growth (p<0.001) and abolished the effect of 10 microM minoxidil, diazoxide and NNC 55-0118; glibenclamide (10 microM) had no effect, but prevented stimulation by 10 microM minoxidil. Phenol red stimulated growth (p<0.001), but channel modulator responses remained unaltered. Thus, deer follicles offer a practical, ethically advantageous in vitro bioassay that reflects clinical responses in vivo. The results indicate direct actions of K(ATP) channel modulators within hair follicles via two types of channels, with SUR 1 and SUR 2, probably SUR2B, sulfonylurea receptors.

INTRODUÇÃO: A lesão de isquemia/reperfusão hepática ocorre durante cirurgias hepáticas de grande porte, transplante de fígado e no trauma abdominal. A lesão de isquemia/reperfusão hepática ocasiona lesões no fígado e pode desencadear uma síndrome inflamatória sistêmica com lesões de órgãos a distância. Estudos anteriores demonstraram que o diazóxido protege outros órgãos (coração, rins, cérebro) da lesão de isquemia/reperfusão destes órgãos. OBJETIVO: Investigar o efeito da administração do diazóxido na lesão de isquemia/reperfusão hepática. MÉTODOS: Ratos Wistar machos foram divididos em 3 grupos. Em 2 grupos, os animais foram submetidos à isquemia hepática parcial realizada por clampeamento do pedículo dos lobos mediano e lateral anterior esquerdo durante uma hora sob ventilação mecânica. Grupo Salina (n=26): ratos receberam solução salina e Grupo Diazóxido (n=26): ratos receberam diazóxido EV ( 3.5mg/kg ) 15 minutos antes da reperfusão hepática. No terceiro grupo, Grupo Controle (n = 22 ), os ratos foram submetidos apenas à anestesia e manipulação cirúrgica. Quatro e 24 horas após os procedimentos, amostras de sangue foram recolhidas para determinações de AST, ALT, TNF-alfa, IL-6, IL-10, de nitrito/nitrato, creatinina. Amostras teciduais do fígado foram analisadas para dosagem do malondialdeído (MDA), para o estudo das funções oxidativas e fosforilativas mitocondriais, e para a análise histológica. Pela coleta de tecido pulomonar, a permeabilidade vascular pulmonar e a atividade da mieloperoxidade (MPO) também foram determinados. RESULTADOS: Quatro horas após, a reperfusão o Grupo Diazóxido apresentou elevações de AST, ALT, TNF-alfa, IL-6, IL-10 e níveis séricos de nitrito/nitrato significativamente menores que o Grupo Controle (p < 0,05). Observou-se uma redução significativa da disfunção mitocondrial hepática no Grupo Diazóxido em comparação com o Grupo Controle (p < 0,05). Não foram observadas diferenças no conteúdo de MDA fígado, na creatinina sérica e na permeabilidade vascular pulmonar, e na atividade da MPO entre os grupos. Vinte e quatro horas após, a reperfusão o Grupo Diazóxido registrou uma redução de AST, ALT quando comparado ao Grupo Controle (p < 0,05). CONCLUSÃO: O Diazóxido mantém a função mitocondrial do fígado, aumenta a tolerância fígado à lesão de Isquemia/Reperfusão e reduz a resposta inflamatória sistêmica. Esses efeitos requerem comprovações adicionais para o uso na prática clínica / INTRODUCTION: Significant liver ischemia/reperfusion injury can occur during hepatic surgeries, liver transplantation and abdominal trauma. Hepatic ischemia/reperfusion can trigger a local and systemic inflammatory syndrome. Previous studies have shown that diazoxide protects other organs (heart, kidneys, brain) from ischemia/reperfusion injury. AIM: To investigate the effect of diazoxide administration on liver ischemic/reperfusion injury. METHODS: Wistar male rats were divided into 3 groups. In two groups the rats underwent partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral segments during an hour under mechanical ventilation. Saline Group (n=26): rats received saline and Diazoxide Group (n=26): rats received IV diazoxide (3.5mg/kg) 15 minutes before liver reperfusion. The third group, the Control Group (n=22) the rats underwent only anesthesia and surgical manipulation. Four and 24 hours after the procedure blood were collected for determinations of AST, ALT, TNF-alfa, IL-6, IL-10, nitrite/nitrate, creatinine. Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidade (MPO) were also determined. RESULTS: Four hours after reperfusion Diazoxide Group presented elevation of AST, ALT, TNF-alfa, IL-6, IL-10 and nitrite/nitrate serum levels significantly lower than Control Group (p < 0.05). A significant reduction on liver mitochondrial dysfunction were observed in Diazoxide Group compared to Control Group (p < 0.05). No differences in liver MDA content,serum creatinine and in pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion Diazoxide Group showed a reduction of AST, ALT and TGF?1 serum levels when compared to Control group (p < 0.05). CONCLUSION: Diazoxide maintains liver mitochondrial function, increases liver tolerance to I/R injury, and reduces systemic inflammatory response. These effects require further evaluations for using in a clinical setting

<p>Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca²⁺]_i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from <i>ob/ob</i>-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K⁺ channels (K_ATP channels), depolarization, and Ca²⁺ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca²⁺]_i. When [Ca²⁺]_i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca²⁺]_i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca²⁺]_i oscillations are present. By permanently opening or closing the K_ATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca²⁺]_i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca²⁺]_i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca²⁺]_i, acting in synergy.</p>

Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca2+]i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from ob/ob-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (KATP channels), depolarization, and Ca2+ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca2+]i. When [Ca2+]i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca2+]i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca2+]i oscillations are present. By permanently opening or closing the KATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca2+]i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca2+]i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca2+]i, acting in synergy.

INTRODUÇÃO: A lesão de isquemia/reperfusão hepática ocorre durante cirurgias hepáticas de grande porte, transplante de fígado e no trauma abdominal. A lesão de isquemia/reperfusão hepática ocasiona lesões no fígado e pode desencadear uma síndrome inflamatória sistêmica com lesões de órgãos a distância. Estudos anteriores demonstraram que o diazóxido protege outros órgãos (coração, rins, cérebro) da lesão de isquemia/reperfusão destes órgãos. OBJETIVO: Investigar o efeito da administração do diazóxido na lesão de isquemia/reperfusão hepática. MÉTODOS: Ratos Wistar machos foram divididos em 3 grupos. Em 2 grupos, os animais foram submetidos à isquemia hepática parcial realizada por clampeamento do pedículo dos lobos mediano e lateral anterior esquerdo durante uma hora sob ventilação mecânica. Grupo Salina (n=26): ratos receberam solução salina e Grupo Diazóxido (n=26): ratos receberam diazóxido EV ( 3.5mg/kg ) 15 minutos antes da reperfusão hepática. No terceiro grupo, Grupo Controle (n = 22 ), os ratos foram submetidos apenas à anestesia e manipulação cirúrgica. Quatro e 24 horas após os procedimentos, amostras de sangue foram recolhidas para determinações de AST, ALT, TNF-alfa, IL-6, IL-10, de nitrito/nitrato, creatinina. Amostras teciduais do fígado foram analisadas para dosagem do malondialdeído (MDA), para o estudo das funções oxidativas e fosforilativas mitocondriais, e para a análise histológica. Pela coleta de tecido pulomonar, a permeabilidade vascular pulmonar e a atividade da mieloperoxidade (MPO) também foram determinados. RESULTADOS: Quatro horas após, a reperfusão o Grupo Diazóxido apresentou elevações de AST, ALT, TNF-alfa, IL-6, IL-10 e níveis séricos de nitrito/nitrato significativamente menores que o Grupo Controle (p < 0,05). Observou-se uma redução significativa da disfunção mitocondrial hepática no Grupo Diazóxido em comparação com o Grupo Controle (p < 0,05). Não foram observadas diferenças no conteúdo de MDA fígado, na creatinina sérica e na permeabilidade vascular pulmonar, e na atividade da MPO entre os grupos. Vinte e quatro horas após, a reperfusão o Grupo Diazóxido registrou uma redução de AST, ALT quando comparado ao Grupo Controle (p < 0,05). CONCLUSÃO: O Diazóxido mantém a função mitocondrial do fígado, aumenta a tolerância fígado à lesão de Isquemia/Reperfusão e reduz a resposta inflamatória sistêmica. Esses efeitos requerem comprovações adicionais para o uso na prática clínica / INTRODUCTION: Significant liver ischemia/reperfusion injury can occur during hepatic surgeries, liver transplantation and abdominal trauma. Hepatic ischemia/reperfusion can trigger a local and systemic inflammatory syndrome. Previous studies have shown that diazoxide protects other organs (heart, kidneys, brain) from ischemia/reperfusion injury. AIM: To investigate the effect of diazoxide administration on liver ischemic/reperfusion injury. METHODS: Wistar male rats were divided into 3 groups. In two groups the rats underwent partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral segments during an hour under mechanical ventilation. Saline Group (n=26): rats received saline and Diazoxide Group (n=26): rats received IV diazoxide (3.5mg/kg) 15 minutes before liver reperfusion. The third group, the Control Group (n=22) the rats underwent only anesthesia and surgical manipulation. Four and 24 hours after the procedure blood were collected for determinations of AST, ALT, TNF-alfa, IL-6, IL-10, nitrite/nitrate, creatinine. Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidade (MPO) were also determined. RESULTS: Four hours after reperfusion Diazoxide Group presented elevation of AST, ALT, TNF-alfa, IL-6, IL-10 and nitrite/nitrate serum levels significantly lower than Control Group (p < 0.05). A significant reduction on liver mitochondrial dysfunction were observed in Diazoxide Group compared to Control Group (p < 0.05). No differences in liver MDA content,serum creatinine and in pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion Diazoxide Group showed a reduction of AST, ALT and TGF?1 serum levels when compared to Control group (p < 0.05). CONCLUSION: Diazoxide maintains liver mitochondrial function, increases liver tolerance to I/R injury, and reduces systemic inflammatory response. These effects require further evaluations for using in a clinical setting

Abstract Heart muscle has to work constantly throughout the life and its energy metabolism is heavily dependent on a continuous supply of oxygen. Energy metabolism must be effectively regulated to meet the demands of changing workloads in different circumstances. If the oxygen supply is interrupted, the function of the heart is easily disturbed and cells injured. Calcium metabolism is of great importance in these pathological conditions. In this thesis respiratory regulation was studied by non-destructive optical methods in mouse heart. The myoglobin-deficient mouse was used as an experimental model to avoid the artefact caused by intracellular myoglobin. Results show that increased consumption of energy and oxygen lead to concomitant reduction of cytochrome aa3 and oxidation of flavoproteins. This finding supports the view that cell respiration in intact myocardium is dominantly regulated at the level of the respiratory chain. The intracellular Ca2+ accumulation during ischemia is one of the major causes of irreversible ischemia-reperfusion injury. Ischemic preconditioning (IPC) has been shown to protect the heart muscle significantly from ischemic damage. In this thesis Ca2+ accumulation during ischemia and reperfusion was studied in perfused rat heart using Fura-2 as a fluorescent Ca2+ indicator. As there is a significant decrease in intracellular pH during prolonged ischemia, the pH-dependency of Fura-2 signal was taken into account. It was found that IPC attenuates Ca2+accumulation during ischemia and this was connected to a decrease in mitochondrial membrane potential. Both IPC and the pharmacologically induced preconditioning with the mitoKATP opener diaxozide were shown to be associated with increased production of superoxide monitored by means of lucigenin chemiluminescence. The superoxide production correlated with the oxidation-reduction state of flavoproteins. We also describe here a method for measuring of intracellular free Ca2+ in mouse heart during ischemia by simultaneous monitoring of Fura-2 and the pH probe BCECF fluorescence by means of dual wavelength excitation of both probes. The paradoxical decrease of Fura-2 fluorescence during ischemia indicating decreasing intracellular Ca2+ concentration was due to the pH effect on the dissociation constant of the Fura-2-Ca2+ complex. When the pH-dependency of Fura-2 was compensated, an extensive Ca2+ accumulation during ischemia was detected. Much of the previous literature on this subject must be re-evaluated because the pH-dependency of intracellular Ca2+ probes has been largely overlooked.