Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.

Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.

The intracellular Ca2+ handling system in a cardiac myocyte is of crucial importance. It regulates the contraction and relaxation of the myocyte during the excitation-contraction (EC) coupling. A normal intracellular Ca2+ handling system keeps the contraction of the heart orderly, which represents a powerful force to pump blood to the whole body. However, disarrayed or remodelled cellular structure associated with the intracellular Ca2+ handling system at the subcellular level, such as loss of T-tubule network in diseased conditions, may promote abnormal cardiac EC coupling, leading to genesis of cardiac arrhythmias impairing cardiac mechanical functions. Up to date, it is still incompletely understood how the intracellular Ca2+ handling system is altered by changes in subcellular structures of Ca2+ handling systems. In this thesis, biophysically detailed computational models for the intracellular Ca2+ handling system of a cardiac cell were developed, providing a powerful platform to investigate the spatio-temporal complexity associated with the intracellular Ca2+ handling, and its role in generating abnormal cardiac EC coupling. First, a well-validated single cell model was used to investigate how the diastolic and systolic Ca2+ concentration responded to alterations in the model parameters related to the Ca2+ handling system, from which the mechanisms underlying the rate-dependence of EC coupling were analysed. Then, a novel single cell model, with a 2D presentation of the spatial structures of subcellular Ca2+ handling and membrane action potential, of a sheep atrial myocyte was developed for simulating the abnormal intracellular Ca2+ regulation system due to the loss of T-tubules during atrial fibrillation. Variant scenarios of T-tubule loss were considered to investigate the role of the T-tubule in affecting the intracellular Ca2+ regulation. Furthermore, membrane currents' alterations due to the electrical remodelling arising from atrial fibrillation were considered together with the loss of T-tubule. Three typical types of abnormal Ca2+ cycling phenomenon, namely intracellular Ca2+ alternans, spontaneous Ca2+ sparks and intracellular Ca2+ waves were observed in AF conditions. The relationship between T-tubule loss, AF-remodelling and the genesis of delayed afterdepolarizations (DADs) was also investigated. It was shown that the loss of T-tubule in AF condition played an important role in disturbing the Ca2+ regulation system, which increases the risk for a cell to generate impaired contraction.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23834号 / 薬科博第149号 / 新制||薬科||16(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 金子 周司, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Aging is the main risk factor for cognitive decline. The hippocampus, a brain region critical for learning and memory formation, is especially vulnerable to normal and pathological age-related cognitive decline. Dysregulation of both insulin and intracellular Ca2+ signaling appear to coexist and their compromised actions may synergistically contribute to neuronal dysfunction with aging. This dissertation focused on the interaction between insulin, Ca2+ dysregulation, and cognition in hippocampal neurons by examining the contributions of insulin to Ca2+ signaling events that influence memory formation. I tested the hypothesis that insulin would increase cognition in aged animals by altering Ca2+-dependent physiological mechanisms involved in learning. The possible effects of insulin on learning and memory in young and aged rats were studied. In addition, the effects of insulin on the Ca2+-dependent afterhyperpolarization in CA1 pyramidal hippocampal neurons from young and aged animals were compared. Further, primary hippocampal cultures were used to examine the possible effects of insulin on voltage-gated Ca2+ channel activity and Ca2+-induced Ca2+-release; mechanisms known to influence the AHP. We found that intranasal insulin improved memory in aged F344 rats. Young and aged F344 rats were treated with Humalog®, a short-acting insulin analog, or Levemir®, a long-acting insulin analog. The aged rats performed similar to young rats in the Morris Water Maze, a hippocampal dependent spatial learning and memory task. Electrophysiological recordings from CA1 hippocampal neurons revealed that insulin reduced the age-related increase in the Ca2+-dependent afterhyperpolarization, a prominent biomarker of brain aging that is associated with cognitive decline. Patch clamping recording from hippocampal cultured neurons showed that insulin reduced Ca2+ channel currents. Intracellular Ca2+ levels were also monitored using Fura-2 in response to cellular depolarization. Results indicated that a reduction in Ca2+-induced Ca2+-release from intracellular stores occurred in the presence of insulin. These results suggest that increasing brain insulin levels in aged rats may have improved memory by reducing the AHP and intracellular Ca2+concentrations. This study indicates a possible mechanism responsible for the beneficial effects of intranasal insulin on cognitive function absorbed in selective Alzheimer’s patients. Thus, insulin therapy may reduce or prevent age-related compromises to Ca2+ regulatory pathways typically associated with cognitive decline.

Recentemente foi demonstrado que na insuficiência cardíaca (IC), a via final das doenças circulatórias e a maior causa de internação em idosos no Brasil, os danos morfo-funcionais da musculatura esquelética representam um preditor independente de mortalidade. Por outro lado, é conhecido que o treinamento físico aeróbico previne o aparecimento desses prejuízos, que potencialmente podem ter relação com alterações no transporte intracelular de Ca2+. Nesse sentido, o objetivo principal do presente estudo foi avaliar o efeito da IC e do treinamento físico aeróbico na IC em relação à função da musculatura esquelética, à expressão de proteínas que transportam Ca2+ no sóleo e no plantar (DHPRα1, DHPR α2, DHPR β1, RYR, NCX, SERCA 1, SERCA 2, parvalbumina) e à atividade da via proteolítica dependente deste íon nestes músculos (calpaína e calpastatina). Foram utilizados camundongos machos C57B7/6J controle e com inativação dos genes para os receptores α2A e α2C adrenérgicos com 7 meses de idade, quando estes apresentam IC induzida por hiperatividade simpática e 50% de mortalidade. A função muscular foi avaliada pelos testes de deambulação e resistência à inclinação. Tanto a expressão protéica como a atividade proteolítica foram avaliadas por Western blot. Os animais com IC apresentaram disfunção muscular, prejuízos nas proteínas relacionadas ao transiente de Ca2+ tanto no sóleo como no plantar, além de alterações na via proteolítica dependente deste íon em relação aos controle. O treinamento físico, por sua vez, preveniu o aparecimento dessas alterações funcionais e moleculares nos animais com IC. Em conclusão, o treinamento físico aeróbico mostrou-se uma terapia efetiva para a síndrome / Heart failure (HF) is a clinical syndrome with poor prognosis characterized by exercise intolerance, early fatigue and skeletal muscle myopathy, which has been considered an independent predictor of mortality. Conversely, aerobic exercise training prevents skeletal muscle dysfunction, which might be related to altered intracellular Ca2+ handling. Therefore, we tested whether HF would lead to alterations in skeletal musculature function related to changes in Ca2+ handling proteins expression (DHPRα1, DHPR α2, DHPR β1, RYR, NCX, SERCA 1, SERCA 2, parvalbumin) and activity of the Ca2+-dependent proteolysis (calpain and calpastatin) in soleus and plantaris muscles. The potential role of exercise training in preventing Ca2+ handling alterations was also studied. Male wild type and α2A e α2C adrenoceptor knockout (KO) mice on a C56BL/6J genetic background were studied at 7 months of age, when KO mice display HF and skeletal muscle myopathy associated with sympathetic hyperactivity and 50% of mortality. KO mice displayed skeletal muscle dysfunction paralleled by altered Ca2+ handling protein expression and Ca2+- induced proteolysis in both soleus and plantaris. Interestingly, exercise training prevented skeletal muscle dysfunction and Ca2+-induced proteolysis in both soleus and plantaris. Collectively, we provide evidence that improved net balance of Ca2+ handling proteins and decreased Ca2+-induced proteolysis upon exercise training is, at least in part, a compensatory mechanism against skeletal muscle myopathy of sympathetic hyperactivity-induced HF

Recentemente foi demonstrado que na insuficiência cardíaca (IC), a via final das doenças circulatórias e a maior causa de internação em idosos no Brasil, os danos morfo-funcionais da musculatura esquelética representam um preditor independente de mortalidade. Por outro lado, é conhecido que o treinamento físico aeróbico previne o aparecimento desses prejuízos, que potencialmente podem ter relação com alterações no transporte intracelular de Ca2+. Nesse sentido, o objetivo principal do presente estudo foi avaliar o efeito da IC e do treinamento físico aeróbico na IC em relação à função da musculatura esquelética, à expressão de proteínas que transportam Ca2+ no sóleo e no plantar (DHPRα1, DHPR α2, DHPR β1, RYR, NCX, SERCA 1, SERCA 2, parvalbumina) e à atividade da via proteolítica dependente deste íon nestes músculos (calpaína e calpastatina). Foram utilizados camundongos machos C57B7/6J controle e com inativação dos genes para os receptores α2A e α2C adrenérgicos com 7 meses de idade, quando estes apresentam IC induzida por hiperatividade simpática e 50% de mortalidade. A função muscular foi avaliada pelos testes de deambulação e resistência à inclinação. Tanto a expressão protéica como a atividade proteolítica foram avaliadas por Western blot. Os animais com IC apresentaram disfunção muscular, prejuízos nas proteínas relacionadas ao transiente de Ca2+ tanto no sóleo como no plantar, além de alterações na via proteolítica dependente deste íon em relação aos controle. O treinamento físico, por sua vez, preveniu o aparecimento dessas alterações funcionais e moleculares nos animais com IC. Em conclusão, o treinamento físico aeróbico mostrou-se uma terapia efetiva para a síndrome / Heart failure (HF) is a clinical syndrome with poor prognosis characterized by exercise intolerance, early fatigue and skeletal muscle myopathy, which has been considered an independent predictor of mortality. Conversely, aerobic exercise training prevents skeletal muscle dysfunction, which might be related to altered intracellular Ca2+ handling. Therefore, we tested whether HF would lead to alterations in skeletal musculature function related to changes in Ca2+ handling proteins expression (DHPRα1, DHPR α2, DHPR β1, RYR, NCX, SERCA 1, SERCA 2, parvalbumin) and activity of the Ca2+-dependent proteolysis (calpain and calpastatin) in soleus and plantaris muscles. The potential role of exercise training in preventing Ca2+ handling alterations was also studied. Male wild type and α2A e α2C adrenoceptor knockout (KO) mice on a C56BL/6J genetic background were studied at 7 months of age, when KO mice display HF and skeletal muscle myopathy associated with sympathetic hyperactivity and 50% of mortality. KO mice displayed skeletal muscle dysfunction paralleled by altered Ca2+ handling protein expression and Ca2+- induced proteolysis in both soleus and plantaris. Interestingly, exercise training prevented skeletal muscle dysfunction and Ca2+-induced proteolysis in both soleus and plantaris. Collectively, we provide evidence that improved net balance of Ca2+ handling proteins and decreased Ca2+-induced proteolysis upon exercise training is, at least in part, a compensatory mechanism against skeletal muscle myopathy of sympathetic hyperactivity-induced HF

Purinergic receptors are membrane ion channels that are activated by extracellular ATP. In vertebrates, seven genes encode subunits of P2X receptors. The subunits, designated P2X1-7, are 40 - 50% identical in amino acid sequences. P2X receptors are composed of three subunits and are found as homo- and heterotrimers in tissues of vertebrates. P2X receptors have a wide distribution in the organism, functional receptors are found in neurons, glial cells, muscle cells and also in nonexcitable tissues as epithelial, endothelial, and in hemopoietic tissue. Purinergic signalling plays an important role in pain transmission, at CNS injury and immune processes. P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N-and C-termini. Each transmembrane domain contains two amino acids conserved across all P2X subunits. In the first transmembrane domain receptor P2X2 are that Gly30 and Tyr43. In previous experiments performed on P2X2 receptor, electrophysiological measurements demonstrated that substitution of conserved Tyr43 in the first transmembrane domain with alanine prolongs the deactivation time of ion channel after agonist wash out. This work is focused on clarifying the role of conserved tyrosine in the process of opening and closing of ion channel of P2X...