Cyclic nucleotides are important inhibitory regulators of platelet function. These second messengers are hydrolysed by cyclic 3' ,5'-nucleotide phosphodiesterases (PDEs). Three PDEs have been detected in human platelets: cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMP-binding, cGMP-selective phosphodiesterase (PDE5). This research investigates the contribution of PDE2 to the regulation of platelet cyclic nucleotide concentrations, and the effects that PDE2 activity has on the inhibition by cAMP and cGMP of platelet aggregation in response to thrombin or collagen. Increases in platelet cAMP were initiated by stimulation of adenylyl cyclase with prostacyclin (PGI₂), whereas the accumulation of cGMP was induced by nitroprusside (NP). The contributions of PDE2 to the hydrolysis of these cyclic nucleotides were evaluated using a novel inhibitor of the enzyme, 𝘦𝘳𝘺𝘵𝘩𝘳𝘰-9-(2-hydroxy-3-nonyl)adenine (EHNA). Before EHNA was used in experiments on platelet function, its effects on partially purified preparations of the three platelet PDEs were studied. These investigations demonstrated that EHNA is a selective and potent inhibitor of platelet PDE2, and indicated that this compound is a more effective inhibitor of cAMP hydrolysis in the presence than in the absence of cGMP. To measure changes in cyclic nucleotide concentrations, platelets were preincubated with [³H]adenine and ³H]guanine to label the metabolic nucleotide pools. NP caused large concentration-dependent increases in platelet [³H] cGMP levels, and this was associated with highly significant but much smaller increases in [³H] cAMP accumulation, which were optimal with 10 μM NP. Higher concentrations of NP had much less effect on platelet [³H] cAMP. A previous study had shown that the increases in platelet cAMP caused by NP were attributable to the inhibition of PDE3 by cGMP (Maurice and Haslam, 1990a), but the inhibitory component observed with high concentrations of NP had not been explained. The present research showed that the accumulation of cAMP and cGMP induced by high NP concentrations is enhanced by EHNA, and so provides the first demonstration that PDE2 activity restricts NP-induced cyclic nucleotide accumulation. To assess whether these changes in platelet cyclic nucleotide levels were important, platelet aggregation in response to thrombin and collagen was monitored. In these studies, EHNA markedly increased the inhibitory action of NP on platelet aggregation. All the effects of NP on cyclic nucleotide accumulation and on platelet aggregation were blocked by a guanylyl cyclase inhibitor, 1𝘏-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one, confirming that NP acts solely through activation of this enzyme and that the increases in cAMP are secondary to cGMP formation. However, experiments with the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine, which diminished the accumulation of cAMP but not that of cGMP, indicated that the inhibition of platelet aggregation is more closely correlated with the increases in cAMP than with those in cGMP. In experiments in which platelet PDE3 was selectively blocked by lixazinone, the accumulation of [³H]cAMP was greatly increased and a corresponding inhibition of thrombin-induced platelet aggregation was observed. Both of these effects were greatly diminished when PDE2 was stimulated by NP (or cGMP). This research demonstrates for the first time that activation of PDE2 by cGMP has marked effects on platelet function, restricting the inhibition of platelet aggregation by agents that increase platelet cAMP. To investigate the importance of PDE2 in regulating different platelet cAMP levels, the effects of EHNA were studied in the presence of 1 or 20 nM PGI₂. Whereas no significant increase in cAMP accumulation was caused by EHNA in the presence of 1 nM PGI₂, at the higher PGI₂ concentration a marked increase was detected when PDE2 was inhibited. NP potentiated the increase in cAMP seen with low PGI₂ but inhibited that seen with a high PGI₂ concentration, indicating a shift in the relative importance of PDE3 and PDE2 as platelet cAMP was increased. These studies show that in the presence of a high concentration of cAMP alone, or of regulatory cGMP, PDE2 makes a major contribution to the hydrolysis of platelet cAMP. Moreover, the results suggest that PDE2 inhibitors could be of value in the therapeutic modification of platelet responses. / Thesis / Master of Science (MSc)

The type Iα cGMP-dependent protein kinase (PKG Iα) is an essential regulator of vascular tone and systemic blood pressure. Located in the smooth muscle of resistance vessels, PKG Iα stimulates vasodilation through the phosphorylation of multiple intracellular substrates. Its primary regulator is the small molecule, 3',5'-cyclic guanosine monophosphate (cGMP); however, the Iα isoform can also be activated by oxidation. Despite the established physiological importance of PKG Iα, the structural underpinnings of these two activation mechanisms are largely unknown. The work presented in this dissertation demonstrates the importance of the cGMP-binding domain A (CBD-A) in regulating both of these mechanisms of PKG Iα activation. Using a monomeric, N-terminally truncated form of PKG Iα (Δ53), Chapter 2 investigates the mechanism of inhibition through the autoinhibitory domain and the influence of dimerization on cooperative cGMP-dependent activation and cyclic nucleotide selectivity. We observed that autoinhibition occurs in cis, whereas cooperativity requires interprotomer contacts facilitated by the N-terminal dimerization domain. Furthermore, the loss of selectivity for cGMP over cAMP of this construct suggests the dimerization domain plays a critical role in preventing cross-reactivity with cAMP-dependent signaling. These observations culminate into an overarching model wherein binding of cGMP to CBD-A is necessary and sufficient for activation and cooperativity is driven by the dimerization domain. Chapter 3 investigates the cysteine residues that mediate oxidation-dependent activation of PKG Iα. Using PKG Iα constructs with point mutations at specific cysteine residues, it was found that oxidation-dependent activation is driven by C117 in CBD-A. Furthermore, the interprotomer disulfide bond that forms in the dimerization domain at C42 does not contribute to this mechanism. Finally, we propose a model wherein the disulfide bond that forms between C117 and the adjacent cysteine at position 195 acts as a protective mechanism to prevent activation and higher oxidation states form contacts with nearby residues in the linker region of PKG Iα to disrupt binding of the adjacent autoinhibitory domain to the catalytic domain. Finally, Chapter 4 provides a discussion of the results presented herein in context with previous studies and suggests future directions for the PKG field.

No description available.

The eATP pathway begins a cascade of events which includes the involvement of nitric oxide synthase (NOS) and nitrate reductase (NR) in the production of nitric oxide (NO). Research has shown that SNAP (S-nitroso-N-acetylpenicillamine) and NONOates (diazeniumdiolates) promote the availability of NO and, with the addition of guanylate cyclase, form cyclic guanine monophosphate (cGMP), and root hair growth is promoted. Phosphodiesterases (PDE) break down the cGMP and agents such as IBMX and Viagra inhibit the PDEs thereby inhibiting root hair growth. Several questions remain to be answered. How much cGMP is necessary for the promotion of root hair growth? Is there an optimal concentration of cGMP which stimulates root hair growth, above which is inhibitory, or below which is ineffective? Is there a “non-hydrolyzable analog” of cGMP which is more effective at promoting root hair growth? Is it possible to see inhibition of root hair growth with exposure to a known inhibitor, such as ATPγS, and then reverse the inhibition with a “non-hydrolyzable analog” of cGMP? Answering these vi questions is the substance of this research and the answers will provide direction and understanding to the growth-promoting and regulatory role eATP plays in signal transduction pathways in plants. With the hypothesis asking whether the effects of NO on root hair growth is cGMP-dependent or cGMP-independent we found that there is no consistent concentration of non-hydrolyzable cGMP analog which promotes root hair growth. Additionally we found that the 8-Br-cGMP analog promotes root hair growth more consistently in Arabidopsis thaliana than its counterpart, dibutyryl cGMP. We substantiated previously published results showing an inhibition of root hair growth when root hairs were exposed to high concentrations of ATPγS. Based on these results we believe the promotion of root hair growth in Arabidopsis thaliana to be mediated independently of cGMP. / text

Die NO-sensitive Guanylyl-Cyclase (NO-GC) ist der wichtigste Rezeptor für das freigesetzte Signalmolekül NO und katalysiert die Bildung des second Messenger cGMP. Die NO/cGMP Signalkaskade ist im kardiovaskulären System essentiell für die Hemmung der Thrombozytenaggregation und des Tonus der glatten Gefäßmuskulatur und trägt damit zur Regulation des Blutdrucks bei. In der Arbeitsgruppe wurden Mauslinien generiert, bei denen die NO-GC ubiquitär (GCKO) oder spezifisch in glatten Muskelzellen (SM-GCKO) ausgeschaltet ist. Beide Mausstämme zeigen eine arterielle Hypertonie mit einem Anstieg des systolischen Blutdrucks um 30 mmHg im Vergleich zu den jeweiligen Kontrolltieren. Neben cGMP ist auch cAMP als weiterer second messenger in einer Reihe von Regulationsprozessen im kardiovaskulären System involviert. Die Intensität und Dauer eines cAMP-Signals wird zum einen durch seine Synthese durch die Adenylyl-Cyclasen, zum anderen durch seine Hydrolyse durch die Phosphodiesterasen (PDE) bestimmt. Dabei spielt die PDE3 als cGMP-inhibierte, cAMP-abbauende PDE eine wichtige Rolle im sogenannten cGMP/cAMP-Crosstalk. Ein wichtiges Instrument zur Untersuchung PDE3-abhängiger Prozesse ist der spezifische Hemmstoff Milrinon. Innerhalb dieser Arbeit konnte gezeigt werden, dass die PDE3-Expression abhängig ist von der Expression des cGMP-produzierenden Enzyms NO-GC: So zeigte sich in Thrombozyten wie auch in glatten Gefäßmuskelzellen nach Deletion der NO-GC im Vergleich zu den Kontrolltieren eine Reduktion der PDE3 um die Hälfte. Diese Reduktion der PDE3-Expression war sowohl in den glatten Muskelzellen von GCKO wie auch von SM-GCKO-Mäusen zu finden. Weiterhin konnte dargestellt werden, dass die Down-Regulation der PDE3 in glatter Gefäßmuskulatur in SM-GCKO-Mäusen parallel zur Reduktion der NO-GC und nicht parallel zum daraus resultierenden Anstieg des Blutdrucks verläuft. Die Reduktion der PDE3-Expression ging mit einer Verminderung der Aktivität in den Thrombozyten und glatten Muskelzellen des GCKOs einher. In Herzmuskelgewebe dagegen änderten sich Expression und Aktivität der PDE3 nicht. Der spezifische PDE3-Hemmstoff Milrinon führte zu einem weiteren Anstieg des systolischen Blutdrucks in den KO-Linien, nicht aber in Kontrolltieren. Zusammenfassend spielt die PDE3 eine wichtige Rolle im cGMP/cAMP-Crosstalk sowohl in Thrombozyten als auch in glatten Gefäßmuskelzellen von Mäusen. / Role of PDE3 on cGMP/cAMP crosstalk in mice deficient in NO-sensitive guanylyl cyclase

Abstract The rate of hyperlipidemia is increasing due to the change of people¡¦s life style and growing aging problem in recent years. Many studies indicated that hyperlipidemia is the base of atherosclerosis which mainly causes cardiovascular diseases. Therefore, how to prevent the sorts of diseases becomes an important topic for the Department of Health. The main objective of the study is to evaluate marketing strategies of Micronized Fenofibrate 200 mg in the case study company. The study examined the treatments of Micronized Fenofibrate 200 mg in hyperlipidemia and its marketing strategies. The study results may provide advice of decreasing medical expenditure for the Bureau of National Health Insurance. The study mainly applied Market Segmentation, product STP (Segment, Target and Position) strategies and relevant document of marketing strategies. The method is to analyze the case product by using Porter¡¦s 5 forces analysis to describe the product competition market. Via SWOT analysis, the study examined the outside opportunities and threats as well as inside superior and inferior; and then inspected the drug marketing strategies with 4P analysis (Product, Price, Promotion, Place ) . Meanwhile, this study conducted a questionnaire to physicians about the case product. The results were also compared to the drug sell situation and market shares of the case company. The result shows that through definite market segmentation, product STP, professional representative¡¦s promotion and active participation in medical conferences, the case product successfully builds its marketing channel and brand name identity within 6 years. The case product has grown from 300,000 pills in 2001 to more than 6 million pills in 2007. The case company has transformed from traditional pharmaceutical company into specialized diabetic medication company. Hence, building the brand identity, enhancing pharmaceutic technique and clarifying market position may become the main stream of pharmaceutical industry competition in the near future. Key word: drugs for hyperlipidemia, cGMP, marketing strategy

Cells of the cardiovascular system translate incoming extracellular signals from hormones and drugs through binding of cell surface receptors, and activation of intracellular signaling cascades allowing modulation of specific cellular function. cAMP and cGMP are ubiquitous second messengers that activate specific signaling machinery used to promote or inhibit cellular functions such as cell migration, cell adhesion and proliferation. Increases in intracellular cAMP or cGMP levels occurs through activation of adenylyl cyclase (cAMP) or guanylyl cyclase (cGMP) or by inhibition of the cAMP and cGMP hydrolyzing enzymes, cyclic nucleotide phosphodiesterases (PDEs). Cyclic nucleotides achieve signaling specificity through compartmentation, a mechanism allowing effective regulation of cAMP or cGMP signaling in discrete parts of the cell in a spatial and temporal manner. Cells of the cardiovascular system such as platelets, vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs) maintain cyclic nucleotide compartmentation through coordinating signaling complexes containing a cAMP or cGMP effector protein and PDEs. Studies reported in this thesis demonstrate that human platelets, VECs and VSMCs each contain distinct cyclic nucleotide signaling complexes, and that based on their composition and selective subcellular localization, regulate specific cellular functions. In platelets, subcellular localization of PDE5 results in differential regulation of PDE5 and selective regulation of Ca2+ release from endoplasmic reticulum stores, an initial step in platelet aggregation and provides a potential therapeutic target in preventing thrombosis. VECs utilize multiple signaling systems to regulate cellular function including cAMP signaling pathways and modification of phosphatidylinositols. These studies demonstrate that a PDE3B-based signaling complex allows integration of both cAMP and phosphatidylinositol-3-kinase-γ (PI3Kγ) signals resulting in increased cell adhesion and cell spreading. Finally, studies in VSMCs demonstrate that PDE5 localization in cells allows cAMP/cGMP cross talk through PDE5 and PDE3A. These results are discussed in the context of further understanding the role of PDEs in mediating cAMP and cGMP signaling and modulation of cell function in cells of the cardiovascular system. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-06-28 13:31:51.428

No description available.

The introduction of mass spectrometry techniques to the field of biology has made possible the exploration of the proteome as a whole system as opposed to prior techniques, such as anti-body based assays or yeast two-hybrid studies, which were strictly limited to the study of a few proteins at a time. This practice has allowed for a systems biology approach of exploring the proteome, with the possibility of viewing entire pathways over increments of time. In this study, the effect of treating Arabidopsis thaliana suspension culture cells with 3’,5’-cyclic guanosine monophosphate (cGMP), which is a native second messenger, was examined. Samples were collected at four time points and proteins were extracted and enriched for both oxidation and phosphorylation before analysis via mass spectrometry. Preliminary results suggest a tendency towards an increased number of phosphorylated proteins as a result of cGMP treatment. The data also showed a sharp increase in methionine oxidation in response to the treatment, occurring within the first ten minutes. This finding suggests that cGMP may utilize methionine oxidation as a mechanism of signal transduction. As such, this study corroborates a growing body of evidence supporting the inclusion of methionine oxidation in intracellular signaling pathways.

Made available in DSpace on 2017-07-21T14:13:07Z (GMT). No. of bitstreams: 1 Vanessa Kovalski Silverio.pdf: 1423306 bytes, checksum: 68729da0a75b158004d6a33911081b96 (MD5) Previous issue date: 2015-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Septic shock, which is triggered by microbial products, is mainly characterized by an inadequate tissue perfusion caused by a decreased in blood pressure, vascular damage, hyporeactivity to vasoconstrictors and disseminated intravascular coagulation, which can lead to multiple organ dysfunction and death. Nitric oxide (NO) is an important player in the pathogenesis of sepsis and septic shock, having both protective and deleterious effects. Trials of nonselective NOS inhibitors have shown increased mean arterial pressure, but also increased pulmonary artery pressure and reduced cardiac output. Thus, despite the intense research, there was no clinical progress so far. Perhaps this is due to lack of compression of NO signaling pathways. There is a knowledge gap on the role of the NO-guanylate cyclase- guanosine 3 ', 5'-cyclic monophosphate (cGMP) pathway during the early stages of sepsis. Most work has studied the role of cGMP by inhibiting the enzyme responsible for its production (guanylate cyclase), however, in this work we chose to try the increased cGMP levels by inhibiting an enzyme responsible for their degradation, phosphodiesterase 5 (PDE5). In the early stages of shock occurs an intense release of vasoconstrictors agents, which can lead to ischemia damage. In this scenario, cGMP could play a key role counterbalancing these agents and preventing tissue damage. Sildenafil, a type-5 cGMP phosphodiesterase inhibitor, increase GMP at smooth muscle cells and promotes vasodilation .Thus, the purpose of this study was to investigate the effect of treatment with sildenafil in the early stages of sepsis. Male Wistar rats were divided into 2 groups of 16 animals each and submitted to either Cecal Ligation and Puncture (CLP) or the sham procedure. Eight hours after the procedure, the CLP and sham groups were randomly assigned to receive sildenafil (10 mg/kg orally) or vehicle. Twenty-four hours after the CLP or sham procedure, the mean arterial blood pressure, systolic and diastolic blood pressure and vascular reactivity to phenylephrine were evaluated. Additionally, blood samples were collected for measurement of nitric oxide (nitrate and nitrite), urea, creatinine, AST, ALT, lactate and CK.; hematological analyzes were also performed. Statistical significance was analyzed by two-way ANOVA followed by post hoc Bonferroni. The results shows that the CLP was able to reproduce the main characteristics of humans sepsis like as, systemic inflamation, hypotension, reduced response to vasoconstrictors, hypoxia and tissue damage. Interestingly, treatment with sildenafil improved and the organ hypoperfusion at early stages of sepsis reducing organ injury. Thus, phosphodiesterase inhibition may be a useful therapeutic strategy if administered at the proper window of opportunity. / O choque séptico, que é desencadeado por produtos microbianos, é caracterizado principalmente por uma perfusão tecidual inadequada causada pela diminuição da pressão arterial, lesão vascular, hiporreatividade a vasoconstritores e coagulação intravascular disseminada, o que pode levar à disfunção de múltiplos órgãos e morte. O óxido nítrico (NO) tem um importante papel na patogênese da sepse e choque séptico, tendo efeitos tanto protetores como deletérios. Ensaios clínicos com inibidores não seletivos da NOS (óxido nítrico sintase) motraram melhora nos níveis da pressão arterial média, porém houve também um aumento da pressão da artéria pulmonar e redução do débito cardíaco. Assim, apesar da investigação intensa, não houve progressos clínicos até agora. Talvez isso se deve à falta de compreensão do NO e suas vias de sinalização. Há por exemplo uma lacuna sobre o papel da enzima guanilato ciclase solúvel e do segundo mensageiro guanosina 3',5'- monofosfato cíclico (cGMP) durante os momentos iniciais da sepse. A maioria dos trabalhos tem estudado o papel do cGMP através da inibição da enzima responsável pela sua produção (guanilato ciclase), porém, neste trabalho optamos por tentar o aumento dos níveis de cGMP através da inibição de uma enzima responsável pela sua degradação, a fosfodiesterase 5 (PDE5). Nos primeiros estágios de choque ocorre uma intensa liberação de agentes vasoconstritores, o que pode levar a danos por isquemia. Neste cenário, o cGMP pode desempenhar um papel chave para contrabalançar esses agentes e impedir danos nos tecidos. O sildenafil, um inibidor de fosfodiesterase do tipo 5, aumenta cGMP em células do músculo liso promovendo vasodilatação. Assim, o propósito do presente estudo foi investigar o efeito do tratamento com sildenafil nas fases iniciais da sepse. Ratos Wistar machos foram divididos em 2 grupos de 16 animais cada e submetidos ao procedimento CLP (Cecal Ligation and Puncture) ou à falsa cirurgia (sham). Oito horas após o procedimento, os grupos CLP e sham foram aleatoriamente divididos para receber sildenafil (10 mg / kg por via oral) ou veículo. Vinte e quatro horas após o procedimento CLP ou sham, a pressão arterial média, sistólica e diastólica e reatividade vascular à fenilefrina foram avaliadas. Além disso, amostras de sangue foram coletadas para a dosagem de óxido nítrico (nitrato e nitrito), uréia, creatinina, AST, ALT, lactato e CK; análises hematológicas também foram realizadas. A significância estatística foi analisada por ANOVA de duas vias seguida de post hoc Bonferroni. Os resultados mostram que o modelo CLP foi capaz de reproduzir as principais características da sepse como inflamação sistêmica, hipotensão, redução da resposta ao vasoconstritores, hipóxia e dano tecidual. Interessantemente, o tratamento com sildenafil melhorou a hipoperfusão em estágios iniciais de sepse reduzindo a lesão de órgãos. Sendo assim, inibidores das fosfodiasterases, como o sildenafil, podem ser ferramentas terapêuticas úteis para o tratamento da sepse, desde que administrados na correta janela de oportunidade.