Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 104-122).

Thesis (Ph.D) -- Virginia Commonwealth University, 2005. / Title from title-page of electronic thesis. Prepared for: Dept. of Physiology. Bibliography: p. 160-169.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2006. / Vita. Includes bibliographical references (leaves 90-106).

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Abnormal homeostasis of intracellular Ca2+ plays a deleterious role in muscle pathologies by triggering processes that lead to dysfunction and necrotic or apoptotic cell death. One pathology where there is significant Ca2+ induced cell damage is ischemia, which initiates further damage (also mediated by Ca2+) generated by the required treatment process of revascularization; namely ischemia-reperfusion injury. Pharmacological agents used therapeutically for cell protection, especially for cardiac protection in ischemic heart diseases, have only directly targeted one of the elements regulating Ca2+ homeostasis, the L-type Ca2+ channels (calcium channel blockers). Other agents, like beta blockers, indirectly target various elements, including sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and ryanodine receptors (RyRs). However, there are no pharmacological agents that directly and specifically target these two crucial elements required for intracellular SR Ca2+ homeostasis. Dr. Julio A. Copello’s group has previously studied the cardioprotective agent CGP-37157 (CGP), a benzothiazepine (BZT) derivative of the benzodiazepine (BZD) clonazepam. CGP was previously thought to decrease intracellular SR Ca2+ by acting as a blocker of the mitochondrial Na+/Ca2+ exchanger (Omelchenko et al., 2003). They found, however, that CGP also activates RyRs and inhibits the SERCA, which could better explain the SR effects of the drug (Neumann et al., 2011). These results suggest that drugs inducing partial depletion of SR Ca2+ stores could provide cellular protection in stressful circumstances or processes. The aims of the dissertation were organized based on the two processes that cause damage to muscle cells during ischemia: ischemia and subsequent reperfusion (ischemia-reperfusion injury) (Ibanez et al., 2015). Aim one and two focused on drug-protective action during the ischemic event, while aim three focused on drug protective action in the reperfusion (early post-ischemia) process. In the first Aim, experiments were designed to test the hypotheses that RyRs and/or SERCA could also be the target of i) Drugs with structural similarities to CGP (i.e., other BZTs and some BZDs) and ii) Drug known to confer cellular protection under stressful cellular conditions such as antiepileptic agents. We found that some BZTs (K201, CGP analog) and antiepileptic agents (Sipatrigine and Pimozide) demonstrated potential to prevent SR Ca2+ overload by inhibition of SERCA and, in some cases also by inducing mild activation of RyR channels. These results provided potential mechanisms of action for agents with cell protective action: targeting SERCA and preventing Ca2+ overload in pre-ischemia process. From the results of the first aim, K201 had the most significant effects in both SERCA inhibition and RyRs activation. Therefore, Aim 2 experiments focused on exploring with greater detail the action of the compound K201 on RyRs, SERCA and Ca2+ signaling. We found that K201 is a more potent SERCA blocker than RyR agonist and that SERCA inhibition remains under acidosis mimicking ischemic conditions. In Aim 3, the focus was on testing drugs with potential to prevent the overloaded SR from leaking Ca2+ (via RyRs) upon reperfusion. For that, we have examined various classes of organic polycationic agents in their ability to act as fast and reversible RyRs blockers. Currently, no agent with these characteristics is availableas a therapeutic or has been well defined for use as an experimental drug. The membrane permeable cation DHBP was identified as a potent RyR inhibitor with potential for rapid and transient inhibition of spontaneous SR Ca2+ release during reperfusion. In summary, we have defined the ability of some BZTs and antiepileptic agents (K201, CGP analog, Sipatrigine and Pimozide) to prevent/slow down SR Ca2+ overload by inhibition of SERCA, which may play an important role in their mechanisms of cell protection in ischemic events. In the case of BZT, these drugs may help their cause by producing mild activation of RyR2 channels, In addition, we have identify DHBP as a reversible and fast acting RyR inhibitor with potential as template for development of transient inhibitors of spontaneous SR Ca2+ release which may have significant protective action against injury during early reperfusion of the heart.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os Ãcidos graxos insaturados Ãmega-3 (&#969;-3) e Ãmega-9 (&#969;-9) possuem aÃÃo anti-inflamatÃria e antioxidante, enquanto os Ãmega-6 (&#969;-6) sÃo prÃ-inflamatÃrios. Este estudo verificou os efeitos do prÃ-condicionamento com misturas de Ãleos contendo baixa relaÃÃo &#969;-6/&#969;-3 e elevada relaÃÃo &#969;-9/&#969;-6, em modelo experimental de isquemia-reperfusÃo cerebral. Foram utilizados 42 ratos Wistar, divididos em dois grupos: Controle (n=24) e Teste (n=18). O grupo Controle foi subdividido em 4 grupos de 6 animais, cada: Simulado - Ãgua (Sim-Ãgua), Isquemia-ReperfusÃo - Ãgua (IR-Ãgua), Simulado - IsolipÃdico (Sim-IsolipÃdico) e Isquemia-ReperfusÃo - IsolipÃdico (IR-IsolipÃdico). Os animais receberam Ãgua ou uma mistura isolipÃdica com relaÃÃes &#969;-6/&#969;-3 = 8:1 e &#969;-9/&#969;-6 = 0,4:1 por via orogÃstrica, durante sete dias, conforme seus grupos. O grupo Teste foi subdividido em 3 grupos de 6 animais: IR-Mix1, IR-Mix2 e IR-Mix3. Os animais do grupo Teste receberam misturas oleosas com relaÃÃes &#969;-6/&#969;-3 = 1,4:1 e &#969;-9/&#969;-6 = 3,4:1 , diferindo apenas na fonte de &#969;-3: Mix1, contendo o Ãcido &#969;-3 &#945;-linolÃnico; Mix2, contendo os Ãcidos &#969;-3 &#945;-linolÃnico, eicosapentaenÃico e docosaexaenoico, e Mix 3, contendo os Ãcidos &#969;-3 &#945;-linolÃnico e docosaexaenÃico, administradas por via orogÃstrica, durante sete dias. No sÃtimo dia, os animais dos grupos IR-Ãgua, IR-IsolipÃdico, IR-Mix1, IR-Mix2 e IR-Mix3 foram submetidos à isquemia cerebral com oclusÃo bilateral das artÃrias carÃtidas comuns por 1 hora, seguida de reperfusÃo por 3 horas. Os animais dos grupos Sim-Ãgua e Sim-IsolipÃdico foram submetidos à operaÃÃo simulada. Ao final do experimento, todos os animais foram decapitados e seus cÃrebros fatiados para anÃlise histopatolÃgica da Ãrea CA3 do hipocampo. A morte neuronal foi quantificada pela contagem de neurÃnios vermelhos (NV). Constatou-se que a quantidade de NV no grupo IR-Ãgua (36,83  9,79) foi maior (P = 0,0046) que a observada do grupo Sim-Ãgua (17,67  8,48), bem como a quantidade de NV no grupo IR-IsolipÃdico (29,83  12,19) foi maior (P = 0,0459) que a observada no grupo Sim-IsolipÃdico (14,17  11,62). NÃo foi constatada diferenÃa na quantidade de NV entre os grupos Sim-Ãgua (17,67  8,48) e Sim-IsolipÃdico (14,17  11,62), ou entre os grupos IR-Ãgua (36,83  9,79) e IR-IsolipÃdico (29,83  12,19). A quantidade de NV no grupo IR-Mix1 (12,33  6,31) foi menor que a verificada nos grupos IR-Ãgua (36,83  9,79; P < 0,01) e IR-IsolipÃdico (29,83  12,19; P < 0,05). As quantidades de NV nos grupos IR-Mix2 (10,67  2,81) e IR-Mix3 (7,33  6,47) tambÃm foram menores que as verificadas nos grupos IR-Ãgua (36,83  9,79; P < 0,001) e IR-IsolipÃdico (29,83  12,19; P < 0,01). NÃo foram constatadas diferenÃas nas quantidades de NV entre os grupos IR-Mix1 (12,33  6,31), IR-Mix2 (10,67  2,81) e IR-Mix3 (7,33  6,47), entre si. Conclui-se que, independentemente da fonte de &#969;-3, o prÃ-condicionamento com misturas de Ãleos contendo baixa relaÃÃo &#969;-6/&#969;-3 e elevada relaÃÃo &#969;-9/&#969;-6, protege os neurÃnios contra as lesÃes de isquemia-reperfusÃo cerebral em modelo experimental. / Omega-3 (&#969;-3) and omega-9 (&#969;-9) unsaturated fatty acids are anti-inflammatory and antioxidant, while omega-6 (&#969;-6) fatty acids are pro-inflammatory. This study investigated the preconditioning effects of fatty acids mixes with low ratio &#969;-6/&#969;-3 and high ratio &#969;-9/&#969;-6, in a brain ischemia-reperfusion experimental model. Forty-two Wistar rats were aleatory assigned to two groups: Control (n=24) and Test (n=18). Control group was divided into 4 groups, each with 6 animals: Water-Simulated (Water-Sim), Water - Ischemia-Reperfusion (Water-IR), Isolipid-Simulated (Isolipid-Sim) and Isolipid - Ischemia-Reperfusion (Isolipid-IR). The animals received water or a isolipid mix with &#969;-6/&#969;-3 ratio of 8:1 and &#969;-9/&#969;-6 ratio of 0,4:1 by gavage, for 7 days, according to their groups. Test group was divided into 3 groups of 6 animals: Mix1-IR, Mix2-IR, and Mix3-IR. All animals from Test group received oil mixes with &#969;-6/&#969;-3 ratio of 1,4:1 and &#969;-9/&#969;-6 ratio of 3,4:1 , differing only on the &#969;-3 source: Mix1, with &#969;-3 linolenic acid; Mix2, with &#969;-3 linolenic, eicosapentaenoic and docosahexaenoic acids, and Mix 3, with &#969;-3 linolenic and docosahexaenoic acids, by gavage, for 7 days. At the 7th day, animals from Water-IR, Isolipid-IR, Mix1-IR, Mix2-IR, and Mix3-IR groups were subjected to 1-hour brain ischemia by occlusion of both common carotid arteries, followed by a 3-hour reperfusion. Animals from Water-Sim and Isolipid-Sim groups were submitted to a simulated operation. At the end of the experiment, all animals were decapitated and their brains were sliced and sent to histological analysis of the CA3 hippocampal region. Neuronal death was quantified by the red neurons (RN) count. It was found that the number of RN in Water-IR group (36.83  9.79) was higher (P = 0.0046) than the number observed in Water-Sim group (17.67  8.48), and similarly, the number of RN in Isolipid-IR group (29.83  12.19) was higher (P = 0.0459) than the number observed in Isolipid-Sim group (14.17  11.62). There was no difference between the amount of RN from Water-Sim (17.67  8.48) and Isolipid-Sim (14.17  11.62) groups, nor between Water-IR (36.83  9.79) and Isolipid-IR (29.83  12.19) groups. The number of RN in Mix1-IR group (12.33  6.31) was lower than the number seen in Water-IR (36.83  9.79; P < 0.01) and Isolipid-IR (29.83  12.19; P < 0.05) groups. The amounts of RN in Mix2-IR (10.67  2.81) and Mix3-IR (7.33  6.47) groups were also lower than the amounts observed in IR-Water (36.83  9.79; P < 0.001) and IR-Isolipid (29.83  12.19; P < 0.01) groups. There were no differences between the Mix1-IR (12.33  6.31), Mix2-IR (10.67  2.81) and Mix3-IR (7.33  6.47) groups. In conclusion, regardless of the source of &#969;-3, preconditioning with fatty acids mixes with low ratio &#969;-6/&#969;-3 and high ratio &#969;-9/&#969;-6, protects the neurons against brain ischemia-reperfusion injuries in this experimental model.

No description available.

A growing body of evidence has now accumulated supporting the involvement of oxygen-derived free radicals in the development of myocardial ischemia/reperfusion (I/R) injury. We have, therefore, undertaken the present study to examine (1) I/R-related alterations in myocardial antioxidant capacity in pentobarbital anesthetized open-chest rabbits subjected to left circumflex coronary artery ligation followed by reperfusion; (2) the protective effects of pretreatrnent with allopurinol or the 21-aminosteroid U74006F; (3) alternative mechanisms to xanthine oxidase inhibition for allopurinol protection against I/R injury; and (4) the effect of allopurinol treatment on the antioxidant capacity of erythrocytes in pigs used in a heart-lung transplantation study. In the rabbit myocardium, a marked impairment in myocardial antioxidant capacity developed in association with the onset of irreversible injury, as reflected in the enhancement in glutathione (GSH) depletion and formation of thiobarbituric acid-reactive substances (TBARS) following in vitro incubation of tissue homogenate with tert-butylhydroperoxide (TBHP). During the course of post-ischemic reperfusion, the protracted time-course of alterations in antioxidant capacity dissociated them from the early burst of radical formation known to occur at the onset of post-ischemic reperfusion of the myocardium. When the time-dependent changes in functional indices of antioxidant status (TBHP-induced GSH depletion and formation of TBARS) were analysed in relation to activities of antioxidant enzymes, evidence suggestive of functionally relevant impairments in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and glutathione reductase (GRD) activities was found. These results and our demonstration of significant decreases in the activity of GSH-dependent antioxidant enzymes under acidotic conditions suggest that a transient impairment in the functioning of antioxidant enzymes may be involved in triggering irreversible myocardial I/R injury. Repetitive brief episodes of I/R produced a progressive decrease in myocardial ATP levels, which was not associated with any detectable changes in myocardial antioxidant capacity. Ischemic preconditioning produced by brief episodes of I/R did not affect the severity of subsequently induced I/R injury. These results suggest that brief episodes of myocardial ischemia do not produce oxidative tissue damage and the ischemia-induced depletion in myocardial ATP level is at least partially dissociable from the I/R-related impairment in tissue antioxidant capacity. Isolated Langendorff-perfused rabbit hearts subjected to I/R did not show any changes in antioxidant capacity. However, when intact hearts were subjected to ischemia in vivo and a subsequent reperfusion in vitro, an impairment in myocardial antioxidant capacity became apparent. These results suggest that blood elements, possibly activated neutrophils, may be a crucial factor involved in the development of I/R-induced oxidant injury. Chronic allopurinol pretreatment (1 mg/ml in drinking water or approximately 75 mg/kg/day) for 7 days prior to ischemia provided significant protection against I/R-induced alterations in myocardial antioxidant capacity, but not the decrease in tissue ATP levels. This chronic allopurinol regimen was found to enhance myocardial GRD activity in nonischemic tissue. In addition, both allopurinol and oxypurinol inhibited the transition metal ion-catalysed ascorbate oxidation and lipid peroxidation in vitro, likely as a consequence of their metal chelating properties. Similarly, myoglobin-TBHP-catalysed oxidation of uric acid and lipid peroxidation were also suppressed by allopurinol. All these suggest that allopurinol may favorably alter myocardial antioxidant capacity directly by virtue of its transition metal chelating properties and its antioxidant actions in myoglobin-mediated oxidative processes. The acute administration of 21-aminosteroid U74006F (3 mg/kg, i.v) under conditions comparable to those known to protect against trauma-induced damage in the central nervous system failed to reduce manifestations of oxidative injury in rabbit hearts subjected to ischemia and reperfusion. Although reactive oxy-radicals have been implicated in both types of tissue damage, the observed difference in susceptibility to protection by this steroidal antioxidant suggests that the molecular mechanisms involved are not identical. In the heart-lung transplantation study, erythrocytes from allopurinol-treated pigs (given repeatedly at an oral dose of 50 mg/kg) showed a time/dose-dependent increase in antioxidant capacity as reflected in the decrease in malondialdehyde production following in vitro oxidative challenge. The extent of red cell protection in both donor and recipient animals correlated significantly with the functional viability of the transplanted lung tissue, as assessed by tissue water content. These results suggest that the measurement of erythrocyte antioxidant capacity may provide an useful assessment of generalized alterations in tissue antioxidant status produced by pharmacological interventions. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21960号 / 医博第4502号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM