Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 8 uppsatser.

A neurite auto-imune experimental (NAE) é uma polineuropatia desmielinizante monofásica do sistema nervoso periférico (SNP). A NAE é considerada modelo experimental da síndrome de Guillain-Barré (SGB). Por se tratar de uma doença autoimune, pode ser induzida experimentalmente em camundongos geneticamente susceptíveis, através da imunização com componentes da mielina de nervos periféricos. Para a indução da NAE podem ser utilizados P0 e P2, proteínas da mielina do nervo periférico, ou sequências conhecidas de peptídeos dessas proteínas 180-199 e 58-81 respectivamente, consideradas neuritogênicas, ou ainda transferência adotiva de lifócitos T CD4+, oriundas de camundongos previamente imunizados. Para o presente estudo foram utilizados camundongos fêmeas SJL/J nãográvidas, com idade entre 8 e 12 semanas, pesando de 17 a 20 g. Os animais foram divididos em dois grupos: um controle e outro com NAE. 200 µg da sequência de peptídeos 58-81 de P2 emulsificada em 100 µl de adjuvante de Freund completo (AFC) foram injetados por via subcutânea, em quatro locais na região lombar. Para os controles, foi utilizado solução tamponada de fosfato (PBS), emulsionada em AFC desprovida da sequência 58-81 de peptídeos P2, injetada no mesmo local, na mesma quantidade e forma. Cada camundongo tratado com P2 recebeu 200 ng de toxina pertussis em 100 ?L de PBS intraperitonealmente (i.p.) nos dias 0 e 2 pós-imunização (p.i.). No grupo controle, volumes iguais de PBS e toxina pertussis foram administrados pela mesma via sem o peptídeo. Avaliações da motricidade foram realizadas diariamente até o 60º dia, além de análises funcionais e eletroneuromiográficas. Foram encontradas alterações exclusivamente eletrofisiológicas, desmielinizantes e axonais, em cerca de dois terços dos camundongos. Sendo o camundongo SJL/J considerado o camundongo mais susceptível para a provocação de NAE, os achados do presente estudo indicam a limitação do modelo: ausência de alterações motoras detectáveis clinicamente, ocorrendo distúrbios eletrofisiológicos em apenas parte dos animais. O melhor modelo de NAE continua sendo o provocado no rato Lewis por proteína da mielina periférica bovina. É desejável que se continue buscando modelo experimental de NAE em camundongos, tendo em vista que essa espécie animal é a mais bem estudada na Biologia animal e, por essa razão, dela haver extensa variedade de imunobiológicos disponíveis para estudo da patogenia e fisiopatologia de doenças auto-imunes. / Experimental autoimmune neurits (EAN) is a monophasic demyelinating disease of the peripheral nervous system (PNS). EAN is considered to be the experimental model for Guillain-Barré syndrome (GBS). EAN can be induced in genetically susceptible mice using peripheral myelin components as immunogens: peripheral nerve myelin proteins P0 or P2; peptides sequences of those proteins considered neuritogenic, or the adoptive transfer of TCD4+ lymphocytes from previously immunized mice. In the present study non-pregnant female SJL/J mice aged 8-12 weeks weighint 17-20 g where used. The experimental group was treated as follows. Peptides sequence of P2 (200 µg) emulsified in complete Freund adjuvant (CFA) (100 µl) injected in four sites at the lumbar paravertebral region s.c. Controls were injected in the same sites with equal quantities of phosphate buffer solution emulsified in CFA without the peptides sequence. Each mouse treated with P2 was also treated with 200 ng pertussis toxin in 100 ?L PBS i.p. at the days 0 and 2 post-immunisation. Controls were injected i.p. with equal volumes of PBS and pertussis toxin free of the peptides sequence. Motor strength, posture and coordination were evaluated daily until the 60th day postinoculation besides eletroneuromyographic (EMG) evaluation on the 10th and 30thy days of some mice. No clinical disturbances were observed and in two thirds of the animals demyelinating and axonal features were detected at the EMG. SJL/J mice are considered the most susceptible mouse strain for NAE induction but the findings of the present study indicates the limitations of the model and its reproducibility. The best NAE model is yet the obtained in Lewis rat. It is important to look for a mouse model of EAN because this animal species is the most studied in the animal Biology. The extensive variety of immunobiologic products from mice allows performing studies on the pathogeny or physiopathology of autoimmune diseases more easily.

Variantes genéticas trombogênicas podem aumentar o risco de eventos adversos em pacientes com coronariopatia crônica. Estudos prévios demonstraram que o Haplótipo H2 do gene do receptor P2Y12 apresenta uma maior agregação plaquetária e está associado com a presença de isquemia arterial periférica. A metaloprotease ADAMTS13 é responsável pela clivagem do fator de von Willebrand e recentemente foi associada com doença isquêmica coronariana. O objetivo deste trabalho foi avaliar o efeito das variantes genéticas funcionais trombogênicas dos Haplótipos H1 e H2 do receptor plaquetário P2Y12 e dos polimorfismos C1342G (Q448E), C1852G (P618A) e C2699T (A900V) da metaloprotease ADAMTS13 em 611 pacientes com doença arterial coronariana multiarterial com função ventricular preservada, acompanhados por um período de 05 anos no ensaio clínico do projeto MASS II (Medical, Angioplasty, or Surgery Study II) em relação aos eventos morte, infarto agudo do miocárdio, angina refratária necessitando um novo procedimento e acidente vascular cerebral. Neste estudo, a avaliação dos Haplótipos H1 e H2 nos pacientes do MASS II não encontrou diferença entre estes haplótipos e os eventos estudados. A análise dos polimorfismos da ADAMTS13 não encontrou associação entre os polimorfismos e os eventos estudados, exceto para a variante genética T2699 (Val900) que está associada com o evento morte (OR: 1,67 95%IC: 1-2,78, p= 0,049) e morte por causa cardiovascular (OR: 2,23 95%IC: 1,2-3,94, p=0,004) e apresenta uma diminuição na sobrevida livre de morte por causa cardíaca para os portadores do genótipo TT relacionado à este polimorfismo. A análise dos haplótipos e das combinações alélicas destes polimorfismos não apresentou associação com eventos ou com a sobrevida livre dos eventos nestes pacientes. / Thrombotic genetic variants could improve the risk of adverse events related to coronary arterial disease (CAD). P2Y12 platelet receptor H2 haplotype showed higher aggregation index and a positive association was described between such genetic variant and peripheral artery disease. DAMTS13 is a metaloprotease responsible to von Willebrand factor cleavage recently found correlated to CAD. We tested the genetic variants P2Y12 receptor H1 and H2 haplotypes and ADAMTS13 polymorphisms C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 611 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function in a follow up period of 05 years. The incidence of the end points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for P2Y12 H1 and H2 haplotypes and ADAMTS polymorphisms. In our study, we did not disclose any association between H1 or H2 haplotype groups regarding the incidence of any of the studied cardiovascular end-points. The association of ADAMTS13 genotypes and cardiovascular events did not showed any association between C1342G (Q448E), C1852G (P618A) variants and cardiovascular end points. Our date provide a strong association between T2699 variant and increased risk to death (OR: 1,67 CI: 1-2,78, p= 0,049) and cardiac death (OR: 2,23 CI: 1,2-3,94, p=0,004) in a population with CAD. The allelic combinations and haplotypes obtained from ADAMTS13 polymorphisms were not associated to cardiac end points and survival differences between MASS II patients.

Quimioreceptores centrais (QC) e periféricos (QP) são células especializadas em detectar alterações de CO2, O2 e H+, e promover ajustes na ventilação e pressão arterial via sistema nervoso central. Avaliamos aqui a ação da sinalização purinérgica em áreas que apresentam essa propriedade (RTN, C1, NTScom e RPa) durante as respostas cardiorrespiratórias promovidas pela ativação dos quimiorreceptores, e a possível participação de astrócitos. Encontramos evidências que receptores P2 modulam a resposta de QC no RTN, enquanto que receptores P2Y1 e receptores glutamatérgicos, modulam a resposta de QP no C1, e que a sinalização purinérgica na região do NTScom ou na região RPa não contribui para resposta de QC. A manipulação farmacológica de astrócitos do RTN com fluorocitrato, mas não da região do NTScom e RPa, produz alterações respiratórias via receptores P2. Nossos achados evidenciam a importância e contribuem para descriminação dos mecanismos de ação da sinalização purinérgica na região bulbo ventrolateral rostral durante a ativação QC e QP. / Central (CC) and peripherals (PC) chemoreceptors are specialized cells to detect changes in CO2, O2 and H+, and produce adjustments in ventilation and blood pressure via the central nervous system. Here we evaluate the action of purinergic signaling in areas with this property (RTN, C1, commNTS, RPA) during the cardiorespiratory responses elicited by activation of chemoreceptors, and a possible role of astrocytes. We found evidence that P2 receptors modulate CC responses in RTN, while P2Y1 and glutamate receptors modulate PC responses in C1, and that the purinergic signaling in the RPa and commNTS region does not contribute to CC responses. The pharmacological manipulation of the RTN astrocytes, but not commNTS or RPa, with fluorocitrate produces respiratory changes via P2 receptors. Our findings show the importance and contribute to discrimination of the mechanisms of purinergic signaling in the rostral ventrolateral medulla during CC and PC activation.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:30:21Z : No. of bitstreams: 1 menezes_mf_me_arafo.pdf: 831526 bytes, checksum: 44cecb40bcf8a2490778eb7d99c815ee (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudos recentes demonstram que os receptores purinérgicos estão presentes no núcleo parabraquial lateral (NPBL), uma estrutura pontina envolvida no controle da ingestão de sódio. No presente estudo, investigamos os efeitos das injeções do , -methyleneadenosine 5 -triphosphate ( , -metileno ATP, agonista dos receptores P2X) sozinho ou combinado com o ácido piridoxalfosfato-6-azofenil-2',4'-disulfônico (PPADS, antagonista dos receptores P2X) ou suramin (antagonista não seletivo dos receptores P2) no NPBL sobre a ingestão de NaCl 1,8% induzida por depleção de sódio. Também investigamos os efeitos da injeção de , -metileno ATP sozinho ou combinado com o PPADS no NPBL sobre a pressão arterial média (PAM) e freqüência cardíaca (FC) em ratos saciados e depletados de sódio. Foram utilizados ratos Holtzman com implante de cânulas implantadas bilateralmente em direção ao NPBL. A depleção de sódio foi induzida pelo tratamento com o diurético furosemida (20 mg/kg do peso corporal) acompanhado de uma dieta deficiente em sódio por 24 horas. As injeções bilaterais de , -metileno ATP (2,0 e 4,0 nmol/0,2 μL) no NPBL aumentaram a ingestão de NaCl 1,8% induzida por depleção de sódio (25,3 ± 0,8 e 26,5 ± 0,9 mL/2 h, respectivamente, vs. salina: 15,2 ± 1,3 mL/2 h). O pré-tratamento com o suramin (2,0 nmol/0,2 μL) ou com o PPADS (4,0 nmol/0,2 μL) no NPBL aboliu os efeitos do , -metileno-ATP na ingestão de NaCl 1,8% (15,2 ± 1,2 e 16,9 ± 0,9 mL/2 h, respectivamente). As injeções de PPADS sozinho no NPBL não alteraram a ingestão de NaCl 1,8% (14,6 ± 0,8 mL/2 h vs. salina: 18,3 ± 1,8 mL/2 h). No entanto, as injeções de suramin sozinho no NPBL quase aboliram a ingestão de NaCl 1,8% (5,7 ± 1,9 mL/120 min, vs. salina: 15,5 ± 1,1 mL/120 min) e aumentaram a ingestão de sacarose 2% somente no tempo de 90 minutos (7,1 ± 1,3 vs. salina: 5,3 ± 0,8 mL/90 min) sem alterar... / Recent studies have shown that purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of , -methyleneadenosine 5 -triphosphate ( , - methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced by 1.8% NaCl intake. We also investigated the effects of , -methylene ATP alone or combined with PPADS injected into the LPBN on mean arterial pressure (MAP) and heart rate (HR) on replete and sodium depleted rats. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20 mg/kg of body weight) followed by 24 h of sodium-deficient diet. Bilateral injections of , -methylene ATP (2.0 and 4.0 nmol/0.2 μL) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3 ± 0.8 and 26.5 ± 0.9 mL/2 h, respectively, vs. saline: 15.2 ± 1.3 mL/2 h). Pre-treatment with suramin (2.0 nmol/0.2 μL) or PPADS (4 nmol/0.2 μL) into the LPBN abolished the effects of , - methylene-ATP on 1.8% NaCl intake (15.2 ± 1.2 and 16.9 ± 0.9 mL/2 h, respectively). Injections of PPADS alone into the LPBN did not change 1.8% NaCl intake (14.6 ± 0.8 ml/2 h vs. saline: 18.3 ± 1.8 mL/2 h). However, injections of suramin alone into the LPBN strongly reduced 1.8% NaCl intake (5.7 ± 1.9 mL/120 min, vs. saline: 15.5 ± 1.1 mL/2 h) and increased the 2% sucrose intake only at 90 min (7.1 ± 1.3 vs. saline: 5.3 ± 0.8 mL/90 min), without changing 24h water deprivation-induced water intake (16.7 ± 1.8 mL/2 h vs. saline: 15.0 ± 2.1 mL/2 h)... (Complete abstract click electronic access below)

Quimioreceptores centrais (QC) e periféricos (QP) são células especializadas em detectar alterações de CO2, O2 e H+, e promover ajustes na ventilação e pressão arterial via sistema nervoso central. Avaliamos aqui a ação da sinalização purinérgica em áreas que apresentam essa propriedade (RTN, C1, NTScom e RPa) durante as respostas cardiorrespiratórias promovidas pela ativação dos quimiorreceptores, e a possível participação de astrócitos. Encontramos evidências que receptores P2 modulam a resposta de QC no RTN, enquanto que receptores P2Y1 e receptores glutamatérgicos, modulam a resposta de QP no C1, e que a sinalização purinérgica na região do NTScom ou na região RPa não contribui para resposta de QC. A manipulação farmacológica de astrócitos do RTN com fluorocitrato, mas não da região do NTScom e RPa, produz alterações respiratórias via receptores P2. Nossos achados evidenciam a importância e contribuem para descriminação dos mecanismos de ação da sinalização purinérgica na região bulbo ventrolateral rostral durante a ativação QC e QP. / Central (CC) and peripherals (PC) chemoreceptors are specialized cells to detect changes in CO2, O2 and H+, and produce adjustments in ventilation and blood pressure via the central nervous system. Here we evaluate the action of purinergic signaling in areas with this property (RTN, C1, commNTS, RPA) during the cardiorespiratory responses elicited by activation of chemoreceptors, and a possible role of astrocytes. We found evidence that P2 receptors modulate CC responses in RTN, while P2Y1 and glutamate receptors modulate PC responses in C1, and that the purinergic signaling in the RPa and commNTS region does not contribute to CC responses. The pharmacological manipulation of the RTN astrocytes, but not commNTS or RPa, with fluorocitrate produces respiratory changes via P2 receptors. Our findings show the importance and contribute to discrimination of the mechanisms of purinergic signaling in the rostral ventrolateral medulla during CC and PC activation.

No description available.

Variantes genéticas trombogênicas podem aumentar o risco de eventos adversos em pacientes com coronariopatia crônica. Estudos prévios demonstraram que o Haplótipo H2 do gene do receptor P2Y12 apresenta uma maior agregação plaquetária e está associado com a presença de isquemia arterial periférica. A metaloprotease ADAMTS13 é responsável pela clivagem do fator de von Willebrand e recentemente foi associada com doença isquêmica coronariana. O objetivo deste trabalho foi avaliar o efeito das variantes genéticas funcionais trombogênicas dos Haplótipos H1 e H2 do receptor plaquetário P2Y12 e dos polimorfismos C1342G (Q448E), C1852G (P618A) e C2699T (A900V) da metaloprotease ADAMTS13 em 611 pacientes com doença arterial coronariana multiarterial com função ventricular preservada, acompanhados por um período de 05 anos no ensaio clínico do projeto MASS II (Medical, Angioplasty, or Surgery Study II) em relação aos eventos morte, infarto agudo do miocárdio, angina refratária necessitando um novo procedimento e acidente vascular cerebral. Neste estudo, a avaliação dos Haplótipos H1 e H2 nos pacientes do MASS II não encontrou diferença entre estes haplótipos e os eventos estudados. A análise dos polimorfismos da ADAMTS13 não encontrou associação entre os polimorfismos e os eventos estudados, exceto para a variante genética T2699 (Val900) que está associada com o evento morte (OR: 1,67 95%IC: 1-2,78, p= 0,049) e morte por causa cardiovascular (OR: 2,23 95%IC: 1,2-3,94, p=0,004) e apresenta uma diminuição na sobrevida livre de morte por causa cardíaca para os portadores do genótipo TT relacionado à este polimorfismo. A análise dos haplótipos e das combinações alélicas destes polimorfismos não apresentou associação com eventos ou com a sobrevida livre dos eventos nestes pacientes. / Thrombotic genetic variants could improve the risk of adverse events related to coronary arterial disease (CAD). P2Y12 platelet receptor H2 haplotype showed higher aggregation index and a positive association was described between such genetic variant and peripheral artery disease. DAMTS13 is a metaloprotease responsible to von Willebrand factor cleavage recently found correlated to CAD. We tested the genetic variants P2Y12 receptor H1 and H2 haplotypes and ADAMTS13 polymorphisms C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 611 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function in a follow up period of 05 years. The incidence of the end points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for P2Y12 H1 and H2 haplotypes and ADAMTS polymorphisms. In our study, we did not disclose any association between H1 or H2 haplotype groups regarding the incidence of any of the studied cardiovascular end-points. The association of ADAMTS13 genotypes and cardiovascular events did not showed any association between C1342G (Q448E), C1852G (P618A) variants and cardiovascular end points. Our date provide a strong association between T2699 variant and increased risk to death (OR: 1,67 CI: 1-2,78, p= 0,049) and cardiac death (OR: 2,23 CI: 1,2-3,94, p=0,004) in a population with CAD. The allelic combinations and haplotypes obtained from ADAMTS13 polymorphisms were not associated to cardiac end points and survival differences between MASS II patients.

Chronic exposure to arsenic-contaminated drinking water is correlated with obstructive lung disease (i.e. chronic obstructive pulmonary disease (COPD), bronchiectasis), reduced lung function and other respiratory effects (e.g. chronic cough, chest sounds). Researchers have associated arsenic exposure with reduced airway immunity. The airway epithelial innate immune system protects underlying tissue from inhaled particulates and pathogens through a variety of mechanisms. Such defects in innate immunity are associated with chronic bacterial infections and development of obstructive airway diseases, including COPD and bronchiectasis. We hypothesize that arsenic exposure may lead to recurrent lung infection and eventual obstructive lung disease by compromising mechanisms essential in airway innate immunity. In the work presented herein we evaluated the effects of arsenic on airway epithelial barrier properties, wound repair capacity, and signaling pathways essential in innate immunity. We previously published that acute (24 hr) arsenic (0.4-3.9 μM as Naarsenite) slowed wound repair in a human bronchial epithelial cell line (16HBE14o-). In the first study we hypothesized arsenic may be affecting wound repair by altering Ca²⁺ signaling that is important in multiple aspects of wound repair, including cell migration. We found wound-induced Ca²⁺ signaling was largely mediated by paracrine ATP in 16HBE14o- cells, and acute (24 hr) arsenic (0.8, 3.9 μM) exposure reduced ATPmediated Ca²⁺ signaling. We identified functional reductions in the ATP receptors P2Y₂ and P2X₄ following arsenic exposure. Both of these receptors are essential in airway innate immunity (e.g. mucociliary clearance). In the second study we found similar reductions in wound repair capacity and ATP-mediated Ca²⁺ signaling in 16HBE14o cells using a chronic (4-5 week) low-dose (0.13, 0.33 μM) arsenic exposure representative of U.S. drinking water standards. Further, wound-induced Ca²⁺ signaling was reduced in primary cultured tracheal cells derived from mice fed arsenic-free or arsenic-supplemented (50 ppb; 1μM=75 ppb) water for four weeks prior to experimentation. In the last study we demonstrated that the structure and function of the airway epithelial barrier was altered by a five-day exposure of arsenic (0.8, 3.9 μM). We conclude that arsenic at environmentally relevant levels compromises key functions in airway epithelial innate immunity that may underlie development of lung disease.

Lau Chui Pik. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 104-122). / Abstracts in English and Chinese. / Chapter Chapter 1 --- --- Introduction --- p.1 / Chapter 1.1 --- Importance of sinoatrial node in heart functions --- p.3 / Chapter 1.2 --- The importance of Adenosine 5'-triphosphate (ATP) --- p.5 / "ATP as a neurotransmitter, cotransmitter and neuromodulator" --- p.5 / Role of ATP in the heart --- p.7 / Chapter 1.3 --- Importance of delayed rectifier potassium channels (Ik) in the heart --- p.9 / Delayed rectifier potassium channel --- p.10 / Properties of Ik channels in the sinoatrial nodal (SAN)cells --- p.11 / Importance of Ik on heart function --- p.14 / Chapter 1.4 --- Drug/hormone/neurotransmitter modulation of Ik --- p.15 / Drugs modulations of Ik --- p.15 / Hormones/neurotransmitters modulations of Ik --- p.18 / Chapter 1.5 --- Problems encountered in using extracellular ATP on experiments --- p.23 / Chapter 1.6 --- Classification of P2-purinergic receptors --- p.24 / Major nucleotide receptors --- p.24 / p2X receptors --- p.26 / p2Y receptors --- p.28 / 1.7Objectives of the experiment --- p.30 / Chapter Chapter 2 --- --- Materials & Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Solutions --- p.32 / Chapter 2.1.2 --- Enzymes --- p.34 / Chapter 2.1.3 --- Drugs --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Isolation of guinea pig SAN cells --- p.35 / Chapter 2.2.2 --- Identification of SAN region --- p.36 / Chapter 2.2.3 --- Obtaining of single SAN cells --- p.38 / Chapter 2.2.4 --- Preparation of micro-pipettes --- p.40 / Chapter 2.2.5 --- The Patch Clamp Technique --- p.40 / Recording configurations --- p.41 / Electrical recordings --- p.44 / Formation of gigaseal on cell membrane and the development of whole-cell configuration --- p.45 / The changing of bathing solution and addition of drugs --- p.46 / The voltage clamp protocol --- p.47 / Data acquisition and analysis --- p.48 / Statistics --- p.48 / Chapter Chapter 3 --- --- Results --- p.49 / Chapter 3.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.50 / Chapter 3.1.1 --- Characterization of IKs currents --- p.50 / Chapter 3.1.2 --- Stimulatory effect of extracellular A TP on IKs current --- p.51 / Chapter 3.1.3 --- Current-Voltage relationship of ATP on IKs current --- p.57 / Chapter 3.1.4 --- Percentage increase of IKs current in the presence of different [ATP] o --- p.63 / Chapter 3.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.71 / Chapter 3.2.1 --- Effect of 100 μMATP-γS and adenosine on IKs --- p.71 / Chapter 3.2.2 --- Percentage increase of IKs in the presence of adenosine and ATP-γS --- p.76 / Chapter 3.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.80 / Chapter 3.3.1 --- Effects of GTP-γS alone on IKs --- p.80 / Chapter 3.3.2 --- Effect of 100 μM ATP in the presence of GTP-yS on IKs --- p.83 / Chapter Chapter 4 --- --- Discussion --- p.86 / Chapter 4.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.87 / Chapter 4.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.92 / Chapter 4.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.97 / Chapter 4.4 --- Limitations of this study --- p.102 / Chapter 4.5 --- Future studies --- p.102 / Chapter Chapter 5 --- --- References --- p.104