The sodium-calcium exchanger (NCX) is a countertransporter of Na+ and Ca2+ across most cell membranes. It has been identified as an essential component of Ca2+ homeostasis in physiological and disease conditions in both cardiovascular and neurological settings. The exchanger not only transports Na+ and Ca2+, but is also regulated by these ions. Although ionic regulatory profiles differ between NCX isoforms, similar regulatory domains have been identified. Previous structure-function studies have determined key residues within these domains, particularly in the eXchanger Inhibitory Peptide region (XIP) and the Ca2+ binding domains (CBD1/2), which have a direct impact on ionic regulation of the outward exchange currents. Recent structural studies of the Ca2+ binding domains of NCX1 suggest a mechanism by which Ca2+ binding would not only be essential for activation of current but may also influence Na+-dependent inactivation. The alternative splice region is located within the Ca2+ binding domain and may play a role in mediating these regulatory phenotypes. Previous studies have demonstrated that specific combinations of the mutually-exclusive and cassette exons are associated with profound effects on ionic regulation in NCX1. This study focuses on examining the mechanisms by which the alternative splice region, in combination with specific regulatory domains, modulates exchange activity in two isoforms, NCX1 and NCX2. Chimaeric and mutant constructs in the alternative splice region were expressed in Xenopus oocytes and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. Substitution of the region corresponding to the mutually exclusive exon in either exchanger greatly reduced the extent of Na+-dependent inactivation, independently of intracellular Ca2+ concentrations. However, replacement of both the region corresponding to the mutually exclusive exon A and the XIP region reestablishes a wild-type profile in NCX2. The first mutually exclusive exon is therefore critical in determining Na+ and Ca2+-dependent regulatory properties. Furthermore, non-conserved residues within the XIP region may be essential in maintaining the structural stability of the Na+-dependent inactive state of NCX1, and by interacting with the mutually exclusive exon, may contribute to the structure-function relationship and the distinct regulatory phenotype of each Na+-Ca2+ exchanger variant and isoform.

The sodium-calcium exchanger (NCX) is a countertransporter of Na+ and Ca2+ across most cell membranes. It has been identified as an essential component of Ca2+ homeostasis in physiological and disease conditions in both cardiovascular and neurological settings. The exchanger not only transports Na+ and Ca2+, but is also regulated by these ions. Although ionic regulatory profiles differ between NCX isoforms, similar regulatory domains have been identified. Previous structure-function studies have determined key residues within these domains, particularly in the eXchanger Inhibitory Peptide region (XIP) and the Ca2+ binding domains (CBD1/2), which have a direct impact on ionic regulation of the outward exchange currents. Recent structural studies of the Ca2+ binding domains of NCX1 suggest a mechanism by which Ca2+ binding would not only be essential for activation of current but may also influence Na+-dependent inactivation. The alternative splice region is located within the Ca2+ binding domain and may play a role in mediating these regulatory phenotypes. Previous studies have demonstrated that specific combinations of the mutually-exclusive and cassette exons are associated with profound effects on ionic regulation in NCX1. This study focuses on examining the mechanisms by which the alternative splice region, in combination with specific regulatory domains, modulates exchange activity in two isoforms, NCX1 and NCX2. Chimaeric and mutant constructs in the alternative splice region were expressed in Xenopus oocytes and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. Substitution of the region corresponding to the mutually exclusive exon in either exchanger greatly reduced the extent of Na+-dependent inactivation, independently of intracellular Ca2+ concentrations. However, replacement of both the region corresponding to the mutually exclusive exon A and the XIP region reestablishes a wild-type profile in NCX2. The first mutually exclusive exon is therefore critical in determining Na+ and Ca2+-dependent regulatory properties. Furthermore, non-conserved residues within the XIP region may be essential in maintaining the structural stability of the Na+-dependent inactive state of NCX1, and by interacting with the mutually exclusive exon, may contribute to the structure-function relationship and the distinct regulatory phenotype of each Na+-Ca2+ exchanger variant and isoform.

Hintergrund: Das Vorhofflimmern ist die häufigste Rhythmusstörung des Menschen. Eine gesteigerte Expression des Na+/Ca2+-Austauschers (NCX) ist dabei eine von zahlreichen elektrischen Remodeling-Mechanismen, die bei diesem Krankheitsbild beobachtet werden. Der unabhängige, also in Abwesenheit anderer Remodeling-Mechanismen bestehende Einfluss einer gesteigerten NCX-Aktivität auf die atriale Proarrhythmie ist allerdings immer noch unzureichend untersucht. Der zugrundeliegende Mechanismus des Vorhofflimmerns basiert zumindest teilweise auf einer spontanen atrialen Aktivität in Form der Ausbildung von spontanen Aktionspotentialen (sAPs). Dabei führt eine spontane Ca2+-Freisetzung aus dem sarkoplasmatischen Retikulum (SR) zu einer NCX-vermittelten Ca2+-Extrusion, die einen von NCX getragenen Einwärtsstrom (INCX) genereriert, welcher wiederum eine Depolarisation des Membranpotentials (DAD, delayed afterdeplorazition) nach sich zieht. Diese kann, sofern sie das kritische Membranpotential zur Reaktivierung des Natrium-Stroms (INa) überschreitet, ein sAP induzieren, welches letztendlich das Vorhofflimmern auslösen kann. Methoden: In der vorliegenden Arbeit wurden isolierte atriale Herzmuskelzellen von homozygoten NCX-Überexpressor (OE) und heterozygoten NCX-Knockout (KO) Mäusen und deren korrespondierenden Wildtypen (WTOE und WTKO) mit der Ca2+-imaging- und der patch-clamp-Methode, sowie mit molekularbiologischen und histologischen Methoden untersucht. Ergebnisse: Eine gesteigerte bzw. verringerte atriale NCX-Expression und –Aktivität wurde mithilfe eines Immunoblots, durch eine veränderte NCX-vermittelte Ca2+-Extrusionskapazität und durch Messungen des INCX bestätigt. Das Vorhofgewicht, die Zellgröße, die elektrische Zellkapazität und Fibrosefärbungen ergaben keinen Anhalt für ein strukturelles Remodeling in den untersuchten Mauslinien. Es gab weder Unterschiede in der Anzahl von spontanen Ca2+-Freisetzungen aus dem SR noch in der SR-Ca2+-Beladung. Die Anzahl von DADs, die während eines proarrhythmischen Stimulations-Protokolls auftraten, war im OE im Vergleich zum WTOE und im KO im Vergleich zum WTKO unverändert. Allerdings wurden DADs im OE im Vergleich zum WTOE signifikant häufiger in sAPs übersetzt. Komplementär dazu wurden im KO im Vergleich zum WTKO DADs tendenziell aber statistisch nicht signifikant seltener in sAPs übersetzt. Unter Verwendung eines aggressiveren proarrhythmischen Protokolls in KO und WTKO zeigte sich eine signifikant geringere Übersetzung von DADs in sAPs. Im OE zeigte sich eine erhöhte DAD-Amplitude im Vergleich zum WTOE und komplementär dazu eine Verringerung der DAD-Amplitude im KO im Vergleich zum WTKO. Messungen der L-Typ Ca2+-Ströme (ICa,L) zeigten einen vergrößerten Strom im OE und einen verringerten im KO, bezogen auf WTOE bzw. WTKO. Versuche mit einem Ca2+-Chelator ließen auf eine direkte funktionelle Interaktion zwischen NCX und L-Typ Ca2+-Kanälen (LTCC) schließen. Der K+-Strom (IKtot) war im OE verstärkt und im KO verringert im Vergleich zu den korrespondierenden Wildtypen. Schlussfolgerung: In atrialen murinen Herzmuskelzellen führte eine gesteigerte NCX-Aktivität zu einer erhöhten Anfälligkeit gegenüber sAPs, während eine verminderte NCX-Aktivität zu einem 101 Schutz vor dem Auftreten von sAPs führte. Die vorliegende Arbeit legt die folgenden Mechanismen nahe: Eine gegebene spontane Ca2+-Freisetzung induziert über NCX eine DAD, wobei die Amplitude der DAD direkt abhängig von der NCX-Aktivität ist. Sobald eine DAD die Reizschwelle für die Aktivierung von Na+-Kanälen überschreitet, wird ein sAP ausgelöst. Bei, wie in dieser Arbeit beobachtet, unveränderter Anzahl von spontanen Ca2+-Freisetzungen und damit auch von DADs, machte daher im OE die höhere DAD-Amplitude das Überschreiten der spannungsabhängigen Reizschwelle von sAPs wahrscheinlicher. KO Zellen waren umgekehrt dazu durch den komplementären Mechanismus geschützt, da eine geringere DAD-Amplitude seltener die Schwelle zur Induktion eines sAPs erreichte. Die in dieser Arbeit beschriebene Interaktion zwischen NCX und ICa,L kann als additiver Mechanismus interpretiert werden, durch den im OE bei höherer NCX-Aktivität eine leichtere Reaktivierung von ICa,L erfolgt, was die Induktion eines sAPs möglicherweise zusätzlich unterstützt. Die Befunde der vorliegenden Arbeit zeigen, dass die NCX-Aktivität als unabhängiger Proarrhythmiefaktor, zumindest in den in dieser Arbeit untersuchten Modellen, die Entstehung von atrialen sAPs beeinflussen kann. Insbesondere die Befunde im KO lassen eine Inhibition von NCX zur Behandlung des Vorhofflimmerns als zukünftige experimentelle Therapiestrategie möglich erscheinen.

Drosophila larval neuromuscular junctions (NMJs) serve as a model for synaptic physiology. The molecular sequence of the postsynaptic glutamate receptors has been described; however, the pharmacological profile has not been fully elucidated. Despite the postsynaptic molecular sequence used to classify the receptors as a kainate subtype, they do not respond pharmacologically as such. Kainate does not depolarize the muscle, but dampens evoked EPSP amplitudes. Quantal responses show a decreased amplitude and area under the voltage curve indicative of reduced postsynaptic receptor sensitivity to glutamate transmission. ATPA, a kainate receptor agonist, did not mimic kainate’s action. The metabotropic glutamate receptor agonist t-ACPD had no effect. Domoic acid, a quisqualate receptor antagonist, blocks the postsynaptic receptors without depolarizing the muscle, which supports the presence of quisqualate subtype receptors. The results suggest a direct postsynaptic action of kainate due to partial antagonist action on the quisqualate receptors. There does not appear to be presynaptic auto-regulation via a kainate receptor subtype or a metabotropic auto-receptor. A complete pharmacological profiling of the known receptor subtypes at this NMJ has not yet occurred; however, this study aids in furthering the ongoing investigations to provide a clearer picture of pharmokinetic profile and specificity of the receptor subtypes.

Die Regulation der Expression des myokardialen Na+/Ca2+-Austauschers spielt eine entscheidende Rolle in der Genese von kontraktiler Dysfunktion und Arrhythmie bei der Herzinsuffizienz. Ein besseres Verständnis dieser Regulationsprozesse könnte neue Angriffspunkte für Präventions- und Therapiestrategien liefern. In der vorliegenden Arbeit wurden die Effekte von α1-adrenerger-Stimulation und mechanischer Last, auf die mRNA-Transkription des NCX 1-Gens und weiterer Gene des Kalziumtransportes untersucht. Es wurden elektrisch stimulierte (1 Hz, 1,75 mmol/l Ca2+, optimale Vorlast), rechtsventrikuläre Trabekelpräparate des Kaninchens nach 3, 6, und 10 h kontinuierlicher α1-adrenerger Stimulation durch 10 umol/L PE untersucht. Diese Trabekel zeigten eine Abnahme der NCX- mRNA im Vergleich zur Kontrolle (gleiche Bedingungen, kein PE) auf 77,5 ± 3,2% nach 10h (p<0,05). Dieser Effekt war sowohl in Anwesenheit des selektiven α1-Rezeptorblocker Prazosin (Praz) (13 umol/L), als auch des PKC-Inhibitors GF 109203X (1 umol/l) nicht zu beobachten. Darüber hinaus konnte keine Regulation des NCX durch PE in nicht gedehnten Trabekeln beobachtet werden. Da vermutet wurde, dass der α1-adrenerge Effekt Ca2+-abhängig sein könnte, wurde die Wirkung erhöhter extrazellulärer Ca2+-Konzentrationen (3 mmol/l) untersucht und eine Abnahme der NCX-mRNA auf 77,4 ± 7,8 % (p<0.02) beobachtet. Des Weiteren wurden in Zellkulturexperimenten über 24 h, isolierte Kardiomyozyten von der Ratte und dem Kaninchen α1-adrenerger Stimulation durch 10 umol/l Phenylephrin ausgesetzt. Die mRNA der Gene von NCX 1, SERCA 2a und BNP wurden mit der real-time PCR bestimmt und gegen das Transkriptionsprodukt des GaPDH-Gens normiert. Bei den Kaninchenmyozyten waren keine signifikante Expressionsänderungen der Gene NCX 1 und SERCA 2a gegenüber der Kontrolle (Kultur ohne PE) zu beobachten, während die mRNA von BNP auf 257,7 ± 47,9% (p<0,03) der Kontrolle anstieg. In Rattenmyozyten wurden signifikante Veränderungen für NCX 1 (117.8 ± 7,9%, p<0,035), SERCA 2a (89,3 ± 3,4%, p<0,012), und BNP (284,2 ± 82,4%, p<0.016) beobachtet. Die genannten Befunde lassen erkennen, dass α1-adrenerge Stimulation die NCX-Transkription in isometrisch kontrahierenden, multizellulären Herzmuskelstreifen bei optimaler Vorlast über die Aktivierung der PKC vermindert. Die verminderte NCX-Transkription scheint durch Kalzium vermittelt und hängt von den Lastbedingungen ab. Die vorliegenden Ergebnisse lassen vermuten, dass endokrine und mechanische Faktoren über eine ineinander greifende intrazelluläre Signalkaskade und Endstrecke, die Regulation der NCX 1-Expression beeinflussen. Unterschiedlich starke Veränderungen dieser Faktoren könnten die unterschiedlichen Phänotypen der Herzinsuffizienz erklären und nach besserem Verständnis neue Möglichkeiten für Prävention und Therapie eröffnen.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A simple method is reported to synthesize an anticancer drug, NCX 4040, conveniently in mild conditions using silicon chemistry. A starting material, 4-hydroxybenzyl alcohol, was silylated selectively first to give t-butyldimethylsilyl 4-hydroxybenzyl ether, which was then converted to NCX 4040 by esterification, desilylation, hydrochlorination and nitration.

<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

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Made available in DSpace on 2016-06-02T19:22:58Z (GMT). No. of bitstreams: 1 5111.pdf: 6171719 bytes, checksum: 0356f236abaa5b549156bc72fde00f70 (MD5) Previous issue date: 2012-09-14 / Universidade Federal de Minas Gerais / Some snakes have the ability to survive long periods without food and are capable to ingest large meal size. The ingestion of proportionally large preys triggers an expressive increase on the oxidative metabolic demand (SDA Specific Dinamic Action) which can become several times higher than the resting metabolic rate. The two described extreme situations might lead to cardiac changes to adapt structure and function in order to afford these two opposite physiological demands. During long food deprivation (Phase III), the depletion of the body structure may affect the heart muscle. That should be reverted to the SDA and prevent an overload on cardiovascular system. The standard cardiac function of South-american Rattlesnake, Crotalus durissus terrificus, was described as well as the changes caused by long term food deprivation and SDA. The importance of sodium-calcium exchanger (NCX), functionality of sarcoplasmic reticulum, extracellular calcium dependence and the effect of adrenergic stimulation were tested in adult animals at 30°C, under three different metabolic states: postabsorptive, SDA peak and food deprivation. The ventricular mass does not change after food deprivation and SDA. The force of contraction was higher in the base of the heart if compared to the apex, but there is no difference among the experimental groups and it is probably reflex of the fiber orientation in each region of the ventricle. The sarcoplasmatic reticulum is functional in all groups, but the dependence of reticular calcium is lower during starvation compared to the other groups. Muscle contraction is mostly supported by the extracellular Ca2+. The NCX have minor contribution to force generation (20%) but has a major role pumping calcium out of the cell (faster than SERCA). The increase in extracellular Ca2+ concentration during digestion can augment twitch force and would represent a contractile advantage to support the increased cardiac work without the development of hypertrophy. The adrenergic stimulation produced sustained increase in Fc for a wide range of stimulation frequencies in all the tested groups. / O coração possui a importante capacidade de se remodelar diante de alterações nas demandas funcionais. Serpentes apresentam a capacidade de ingestão de grandes presas e a capacidade de sobreviver a grandes períodos de privação alimentar. A ingestão de grandes massas de alimento demanda uma elevação metabólica e leva a uma compensação na massa ventricular e um aumento na força de contração do miocárdio, evitando uma sobrecarga no sistema cardiovascular. Durante o jejum ocorre depleção da estrutura corpórea podendo também atingir o músculo cardíaco. Para descrever a função cardíaca da Cascavél Sul-americana, Crotalus durissus terrificus, e as possíveis alterações diante dos extremos metabólicos, foi testada a importância do trocador Na+/Ca2+ (NCX), a funcionalidade do retículo sarcoplasmático (RS), a dependência do Ca2+ extracelular e o efeito da estimulação adrenérgica em animais adultos à temperatura constante de 30°C em período pós-absortivo, pico de SDA e após jejum prolongado. Observou-se que a massa ventricular se mantém estável mesmo durante os extremos metabólicos. Há uma diferença na geração de tensão entre as tiras da base e ápice ventriculares, presente nos 3 grupos alimentares e que pode se dar em função da orientação das fibras nas duas regiões do ventrículo. O teste com rianodina mostrou que o RS é funcional nos 3 grupos porém a dependência do cálcio proveniente do RS é menor após o jejum prolongado quando comparado aos demais grupos. O cálcio extracelular é o principal responsável pela contração do miócito cardíaco e o NCX, que fornece uma pequena parte de cálcio para a contração, é o principal bombeador de cálcio para fora da célula e é mais rápido do que a Ca2+- ATPase do RS. O meio extracelular é a principal fonte de cálcio ativador da contração sendo que o aumento de cálcio circulante disponível para a contração nos animais durante a digestão ofereceria uma vantagem contrátil para este grupo sem necessidade de hipertrofia. A adrenalina produziu um aumento substancial na Fc capaz e ser mantido por diferentes frequências de estimulação nos 3 grupos testados.