Dissertations / Theses on the topic 'NO-cGMP pathway'

Depressive disorders are among the most frequent psychiatric diseases in the Western world with prevalence between 9% and 18%. Poor compliance and inappropriate antidepressant discontinuation invokes long-term morbidity, and appear linked to hippocampal shrinkage. Despite major advances in pharmacological treatment of the illness over the past 3040 years, currently available agents have distinct shortfalls both in clinical efficacy and in maintenance of response. This implies a greater long-term morbidity with significant impact on the patient, the patient's family as well as economic implications to health care managers and providers. The major reason for this state of affairs is our poor understanding of the neurobiology of depression and hence, of antidepressant (AD) action. AD drugs are thus not addressing the crucial neurobiological target underlying the illness, and new strategies and treatments are urgently needed. In recent years, depression has been associated with disturbances in excitotoxic glutamatergic activity, yet this has not been systematically evaluated. While the role of neurotransmitters such as serotonin, noradrenaline and dopamine has been extensively studied, new evidence suggests a role for the unique neurotransmitter nitric oxide (NO). Nitric oxide (NO), is activated by glutamatergic systems in various limbic and other regions of the brain, and has recently also been implicated in anxiety and affective disorders. Of special interest is the putative role of NO in cellular memory, synaptic plasticity and cell survival, all-important processes in the neuropathology and neurodevelopment of depression. Recent clinical studies have provided evidence of the role of the NO-pathway in depression, while preclinical studies have demonstrated the anxiolytic and antidepressant actions of nitric oxide synthase (NOS)-inhibitors. Moreover, NO interacts with other classical transmitters that have a regulatory role on mood, particularly the monoamines, as well as glutamate and gammaaminobutyric acid (GABA). In the current study the role of the NO-cGMP pathway in AD action was investigated, after chronic imipramine (IMI) and after IMI withdrawal, using a learned helplessness paradigm. Behavioural changes, hippocampal NOS activity and cGMP accumulation was determined together with pharmacological manipulation of the NO-cGMP pathway. Chronic IMI, 15 mg/kg/day intraperitoneal (ip) administration induced a pronounced reduction in swim immobility time in the forced swim test (FST), with no effect on horizontal or vertical locomotor activity. These behavioural changes were accompanied by a significant reduction in NOS enzyme activity and cGMP accumulation. In order to confirm the involvement of the NO-cGMP pathway in the AD action of IMI, chronic (3 weeks) IMI treatment was followed by an acute withdrawal of 7 days. Acute withdrawal, after chronic IMI treatment, resulted in a significant increase in swim immobility time and an increase in NOS enzyme activity and cGMP levels. In fact, NOS activity was raised above that of control, not just higher than the effect of chronic IMI. In order to assess the possible role of the NMDA-NO-cGMP pathway in AD withdrawal, the NMDA receptor antagonist, memantine, and the NOS/guanylyl cyclase (GC) inhibitor, methylene blue (MB), were administered during the 7 day IMI withdrawal period. Memantine (5 mg/kg/d ip), during the 7 day IMI withdrawal period, significantly reversed the increase in immobility time evoked after IMI withdrawal. This was accompanied by a significant reduction in NOS enzyme activity and a tendency to decrease cGMP levels. This data confirms that the antidepressant action of IMI, as well as IMI withdrawal, is associated with actions on the NMDA-GIu-NO-cGMP pathway. Particularly. IMI withdrawal evokes an increase in glutamate activity that is responsible for NOS activation. During the 7 day IMI withdrawal period, MB (15 mg/kg/d ip) also significantly reversed the increased immobility time after IMI withdrawal and was accompanied by a tendency to decrease NOS enzyme activity and cGMP levels in the rat hippocampus, however statistical significance was not reached. Although not emphatic, this data implies a possible role of the NO-cGMP pathway in AD action and AD withdrawal. In order to determine whether the observed IMI withdrawal effects on the NO-cGMP pathway may occur through an initial destabilisation in the serotonergic system, the 5-HT2a/2c receptor antagonist, ritanserin (4 mg/kg/d ip), was administered during the IMI withdrawal period. These studies revealed that antidepressant withdrawal evokes an increase in 5-HT2-mediated activity, and that antidepressant-induced NOS activation after withdrawal has its origin in serotonergic hyperactivity. Clearly, this is supportive of a distinct relationship between the NO and serotonergic system in antidepressant response. On its own, ritanserin was found to increase NOS and cGMP levels, yet during IMI withdrawal this response was lost, suggesting that IMI withdrawal alters the response to a 5-HT2a/2c receptor antagonist, which may have major clinical implications. In conclusion, the AD action of IMI, as well as chronic IMI withdrawal, involves actions on the NO-cGMP pathway. Withdrawal of ADS is associated with a loss of AD efficacy together with an increase in release of NO and cGMP. The NMDA antagonist, memantine, and the NOS/GC inhibitor, MB, reversed these responses therefore suggesting that the NMDA-GIu-NO-cGMP pathway may be a new putative target in understanding the neurobiology of AD action. Finally, NOS activation following withdrawal suggest that inappropriate withdrawal during the treatment of depression may mediate neurodegenerative pathology observed in recurrent depression, possibly by severely increased hippocampal NOS activity which is toxic to neurons.
Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

The neurobiology of anxiety-related disorders is associated with impaired neuroplasticity. The glutamate/NO/cGMP pathway has been proposed to play a key role in neuroplasticity and neurodevelopment. It was demonstrated in recent reports that chronic co-administration of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the antimuscarinic agent atropine exerts antidepressive-like activity in rats, and that this effect is related to PDE5 inhibition, with consequent elevation of cGMP levels and enhanced protein kinase G stimulation. The current study investigated possible anxiolytic effects of the chronic co-administration of sildenafil and atropine in stress-sensitive Flinders Sensitive Line (FSL) rats. FSL rats received vehicle control, fluoxetine (15 mg/kg), atropine (1 mg/kg), sildenafil (10 mg/kg) or sildenafil plus atropine via intraperitoneal administration, either acutely 30 minutes prior to testing (acutely) or daily for 14 days (chronically). FRL control rats received only vehicle. Thereafter anxiety-like behaviour was evaluated in the social interaction test (SIT - acute) and elevated plus maze (EPM - acute and chronic). The current study also compared to different ways to score the EPM, namely the percentage time spend in the open arms of the EPM and both the number of full and half body open arm entries, and also implemented defecation on the EPM as a measure of anxiety. Vehicle-treated FSL rats exhibited more anxiety-like behaviour than FRL rats in both the SIT and EPM following acute treatment, and in the EPM following chronic treatment. Acute treatment with fluoxetine exerted anxiogenic activity in the SIT and EPM, but anxiolytic activity following chronic administration, as observed in the EPM. In acute treatments neither sildenafil nor sildenafil plus atropine yielded any significant effects on anxiety-like behaviour. However, following chronic treatment, sildenafil exerted anxiolytic activity in the EPM by increasing the time spend in the open arms (45.72% ± 9.94% vs. 20.80% ± 9.94%, P<0.001). Atropine exerted a small anxiolytic response (30.71% ± 8.40% vs. 20.80 ± 9.94%), whereas atropine co-administration was additive to sildenafil alone and yielded an enhanced anxiolytic effect in the elevated plus maze (59.56% ± 4.95% vs. 20.80% ± 9.94%, P<0.001), relative to vehicle control. The percentage time spend in the open arms was scored in the EPM, the results suggested that the chronic treatment with sildenafil plus atropine exert an anxiolytic-like effect in FSL rats and the number of fecal droppings did not increase which is also an indication of an anxiolytic-like effects of the treatment. The current study demonstrated that the chronic treatment with sildenafil, alone or in combination with atropine, exhibit an anxiolytic-like action in stress-sensitive rats. In addition, the data support the clinical potential of using PDE5 inhibitors as antidepressant and anxiolytic strategy and warrant further investigation. Furthermore the study supports the previously proposed key role of the glutamate/NO/cGMP pathway in the neurobiology of anxiety-like disorders, and as an important target for drug development.
Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011

Posttraumatic stress disorder (PTSD) is a severe anxiety disorder characterised by hypothalamic-pituitary-adrenal (HPA)-axis abnormalities, hyperarousal, anxiety, flashbacks of trauma memories and avoidance. Increasing evidence is now accumulating that the disorder is also associated with shrinkage of the hippocampus and cognitive dysfunction that may have its origin in stress-induced excitotoxicity. Animal studies have indeed highlighted a potential role of the excitotoxic glutamatenitric oxide (NO) pathway in the stress response. Since PTSD appears to be an illness that progresses and worsens over time after an initial severe traumatic event, this study has used an animal model that emphasises repeated trauma to investigate the effect of stress on hippocampal NO synthase (NOS) activity, the release of the nitrogen oxide metabolites of NO (NOx), and also the evoked release of cGMP. Furthermore, the modulation and dependency of these responses on glutamate, NO and cGMP activity using drugs selective for these targets, will also be investigated. Rats (n=10/group) were exposed to repeated stress together with saline or drug administration immediately after the stress procedure and continuing for one week post-stress. The animals were then sacrificed for assay of hippocampal NOS activity, NO, and cGMP accumulation. Animals received either the glutamate-NMDA receptor antagonist, memantine (MEM;5mg/kg ip/d), the neuronal NOS selective inhibitor, 7- nitroindazole monosodium salt (7-NINA;20mg/kg ip/d), the cGMP-specific PDE inhibitor, sildenafil (SIL;10mg/kg ip/d) or the NFkb antagonist, pyrollidine dithiocarbamate (PDTC;70mg/kg ip/d). The latter inhibits the nuclear transcription factor, NFkb, responsible for inducing the expression of iNOS, while it also appears to mediate the glutamatergic actions on NOS expression, Stress significantly increased hippocampal NOS activity, as well as significantly increased hippocampal cGMP and NO, levels. These increases were blocked by pretreatment with either PDTC or 7-NINA, while memantine was without effect. Sildenafil significantly augmented stress induced NO, accumulation, as well as cGMP. although the latter failed to reach significance. 7-NINA and memantine significantly blocked the increase in cGMP evoked by time-dependent sensitisation (TDS)-stress, with PDTC attenuating this response, but not significantly. Additionally, administration of each drug separately for seven days without exposure to stress, did not evoke significant changes in NOx levels, compared to the control group. However, significant increases in cGMP levels, compared to the control group, were found with all four drugs. Repeated trauma therefore activates the NO-cGMP pathway, possibly involving actions on both nNOS and iNOS. The NMDA receptor appears less involved after chronic repeated stress, and may have limited therapeutic implications. Sub-cellular NO-modulation, however, may represent an important therapeutic strategy in preventing the effects of severe stress and in treating PTSD.
Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Universidade do Estado do Rio de Janeiro
Estudos publicados nas duas últimas décadas sugerem um aumento do risco de doença cardiovascular (DCV) em pacientes com periodontite, mas os mecanismos fisiopatológicos dessa associação ainda não estão completamente esclarecidos. Uma vez que foi demonstrado aumento da ativação plaquetária e do estresse oxidativo na periodontite, o objetivo do presente estudo foi investigar a via L-arginina-óxido nítrico (NO)- guanosina monofosfato cíclica (GMPc) e parâmetros de estresse oxidativo em plaquetas de pacientes com periodontite, bem como avaliar o efeito do tratamento periodontal não-cirúrgico nessas variáveis. Um total de 10 pacientes sem periodontite (periodontalmente saudáveis ou com gengivite) e 10 pacientes com periodontite participaram do estudo. A avaliação clínica, laboratorial e experimental foi realizada no início do estudo e 90 dias após realização da terapia periodontal básica (grupo periodontite). A avaliação clínica periodontal incluiu registros de: profundidade de bolsa à sondagem (PBS), nível de inserção (NIC), percentual de placa e percentual de sangramento à sondagem. Os seguintes experimentos foram realizados: influxo de L-arginina; atividade e expressão das enzimas óxido nítrico sintase e da arginase; expressão das enzimas guanilato ciclase solúvel e fosfodiesterase 5; determinação dos níveis intraplaquetários de GMPc; agregação plaquetária; avaliação do estresse oxidativo (atividade oxidante total, atividade das enzimas antioxidantes catalase e da superóxido dismutase - SOD); medição dos níveis de proteína C reativa (CRP) e de fibrinogênio. Os resultados obtidos no início do estudo demonstraram ativação do influxo de L-arginina em plaquetas via sistema y+L nos pacientes com periodontite, bem como concentrações intraplaquetárias de GMPc diminuídas e aumento sistêmico da CRP. Após o tratamento periodontal, observou-se redução do percentual de sítios com PBS ≥ 6 mm, NIC 4-5 mm e NIC ≥ 6 mm, aumento nos níveis de GMPc, para níveis comparáveis aos dos pacientes sem periodontite, acompanhado por uma maior atividade das enzimas antioxidantes SOD e catalase. Os demais parâmetros avaliados não apresentaram alterações significativas tanto pré- quanto pós-tratamento. Esses resultados considerados em conjunto sugerem uma menor biodisponibilidade de NO em plaquetas na periodontite e que o tratamento periodontal não-cirúrgico foi capaz de reverter este quadro por um aumento das defesas antioxidantes. Portanto, alterações na via L-arginina-NO-GMPc e no estresse oxidativo podem levar à disfunção plaquetária, que poderia contribuir para um maior risco de DCV nos pacientes com periodontite.
Studies published over the last two decades have suggested an increase of cardiovascular disease (CVD) risk on periodontitis patients, but the physiopathological mechanisms involved in this association are not yet clear. Since it has been demonstrated an enhancement on both platelet activation and oxidative stress on periodontitis patients, the aim of this study was to investigate the L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway on platelets from periodontitis patients, and the effect of non-surgical periodontal treatment in these variables. A total of 10 patients without periodontitis (periodontal healthy controls or gingivitis patients) and 10 periodontitis patients were included in this study. The clinical, laboratorial, and experimental evaluations were performed at the beginning of the study and 90 days after the basic periodontal therapy (periodontitis group). The clinical periodontal evaluation included the measurements of probing pocket depth (PPD), clinical attachment level (CAL), plaque percentage, and percentage of bleeding on probing. The following experiments were performed: L-arginine influx; nitric oxide synthase and arginase enzymes activity and expression; expression of guanylate cyclase and phosphodiesterase-5 enzymes; measurement of intraplatelet cGMP levels; platelet aggregation; oxidative stress evaluation (total oxidant activity and activity of both antioxidant enzymes catalase and superoxide dismutase SOD); measurement of C reactive protein (CRP) and fibrinogen. The initial results demonstrated an activation of L-arginine influx in platelets from periodontitis patients via y+L system, reduced intraplatelet cGMP levels and increased CRP. After periodontal treatment, it was observed reduction on percentage of sites with PPD ≥ 6 mm, CAL 4-5 mm and CAL ≥ 6 mm, enhancement on cGMP levels, to levels comparables to patients without periodontitis, accompanied by a higher activity of both antioxidant enzymes SOD and catalase. The other evaluated parameters did not showed significant alterations before and after periodontal treatment. The present results suggested a decreased NO biodisponibility in platelets from periodontitis patients and that the non-surgical periodontal treatment was effective to revert this condition, due to an enhancement on antioxidant defence. Therefore, alterations on L-arginine-NO-cGMP pathway and oxidative stress may lead to platelet dysfunction, which could contribute to a higher risk of CVD in periodontitis patients.

Le syndrome métabolique (SMet) est caractérisé par la présence chez le même individu de plusieurs anomalies parmi les suivantes: une adiposité abdominale, une insulino-résistance (IR), une intolérance au glucose, une hypertension artérielle et une dyslipidémie. Des études ont révélé que la modulation de la voie de signalisation NO/GMPc dans le SMet peut exercer des effets métaboliques et cardiovasculaires protecteurs. Nous avons exploré, dans ce contexte, l'effet du mirabegron et du BAY 41-2272, deux molécules connues pour leur capacité à activer la voie NO-GMPc. Nous avons d'abord développé un modèle animal expérimental avec deux facteurs principaux du SMet, la dyslipidémie et l’IR. Nos résultats ont montré qu'après 12 semaines d'alimentation riche en fructose et en graisses (HFFD), le lapin Watanabe (WHHL), un modèle animal de dyslipidémie spontanée, présentait une intolérance au glucose, une IR (test HOMA-IR), une aggravation de la dyslipidémie et une diminution de la contractilité cardiaque (approche ex-vivo). Après 12 semaines de traitement, le mirabegron et le BAY 41-2272 ont prévenu le gain de poids et l’augmentation du taux de TG et ont amélioré la sensibilité à l'insuline, la fonction endothéliale des artères carotides et la fonction cardiaque (mirabegron). Ce travail a permis de mettre en place un modèle expérimental combinant la dyslipidémie et l’IR chez le lapin WHHL. De plus, les résultats ont montré que l'activation à long terme de la voie de signalisation NO-GMPc représente une approche pharmacologique prometteuse dans la gestion des complications métaboliques et cardiovasculaires associées au SMet
Metabolic syndrome (MetS) is characterized by abdominal adiposity, insulin resistance (IR), glucose intolerance, arterial hypertension and dyslipidemia. Experimental studies have revealed that modulation of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathway in MetS can exert protective metabolic and cardiovascular effects. In this regard, we explored the effect of mirabegron and BAY 41-2272, two molecules known for their ability to activate the NO-cGMP pathway. We first developed an experimental animal model with two main components of the MetS, dyslipidemia and IR. Our results showed that after 12 weeks of high-fructose high-fat diet (HFFD) feeding, the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model of spontaneous dyslipidemia, exhibited glucose intolerance, IR (HOMA-IR test), an aggravation in dyslipidemia and a decrease in cardiac contractility (ex-vivo approach). Twelve weeks of mirabegron and BAY 41-2272 treatment prevented weight gain and the increase in TG levels and improved insulin sensitivity, carotid endothelial function, and cardiac function (mirabegron). We were able to develop an experimental model combining dyslipidemia and IR in the WHHL rabbit. Furthermore, our results showed that long-term activation of the NO-cGMP signaling pathway represents a promising pharmacological approach in the management of the MetS and its metabolic and cardiovascular consequences

碩士
高雄醫學大學
藥理學研究所
96
The endothelial dysfunction resulting in vessel contraction observed in hypertension appears to be a consequence of high blood pressure. In normal endothelial cell, activation of endothelial nitric oxide synthase (eNOS), soluble guanylyl cyclase (sGC) and protein kinase G (PKG) resulted in vasodilation and anti-hypertension. Our previous studies have demonstrated that KMUP-1, a unique xanthine and piperazine derivative, activated the NO/ sGC/ cGMP pathway, and could lead to vascular relaxation. We used 8 week-old Spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat for our experimental model. In the present study, the experimental rats were subdivided into five groups：(1) W1 (WKY group 1：control), (2) W2 [WKY group 2：treating with KMUP-1 (10 mg/kg)], (3) S1 (SHR group 1：control), (4) S2 [SHR group 2：treating with KMUP-1 (10 mg/kg)], (5) S3 [SHR group 3：treating with KMUP-1 (30 mg/kg)]. During 28 days of treatments, systolic blood pressure (SBP) was measured weekly to confirm whether KMUP-1 could ameliorate SBP in SHR. Furthermore, we used aorta to check eNOS, sGCα1, PKG protein expression by Western blotting.Our results showed that SBP of SHR elevated more than that of WKY with age. KMUP-1 (10 mg/kg) did not significantly decrease SBP of WKY. However, SBP of SHR by treating with KMUP-1 (10 mg/kg, 30 mg/kg) was significantly decreased as compared with SHR control. Moreover, eNOS, sGCα1 and PKG protein expression in SHR or WKY aorta by treating with KMUP-1 were significantly increased. In conclusion, KMUP-1 could active NO/cGMP pathway to improve SBP of SHR, suggesting that KMUP-1 could be a potential drug for hypertension.    Hypoxia exposure induced impairment in the structure and function of cardiopulmonary circulation. The pathological changes of cardiopulmonary arteries included endothelial injury, vessel remodeling, and contraction. It has been confirmed that hypoxia promoted downregulation of endothelial nitric oxide synthase (eNOS), upregulation of Rho kinase (ROCK) and vascular endothelial growth factor (VEGF) expression resulting in vascular contraction and remodeling to induce pulmonary arterial hypertension. Furthermore, activation of eNOS, soluble guanylyl cyclase (sGC), and protein kinase G (PKG) protein expression resulted in pulmonary arterial vasodilation and anti-remodeling. Previous studies have demonstrated that KMUP-1, a unique xanthine and piperazine derivative, activated the NO/sGC/cGMP pathway, and could lead to vascular relaxation. In the present study, the Wistar rats were subdivided into four groups：(1) Normoxia, (2) Hypoxia (10% O2) for 21 days, (3) Hypoxia (10% O2) + KMUP-1 (5 mg/kg/day) for 21 days, (4) Hypoxia (10% O2) + Sildenafil (5 mg/kg/day) for 21 days. After 21 days of hypoxia, we measured pulmonary arterial pressure to evaluate the development of pulmonary arterial hypertension. Through method of Hematoxylin-Eosin staining, we investigated wall thickness of pulmonary artery and right ventricular hypertrophy. Moreover, molecular mechanism was analyzed by Western blotting and immunohistochemistry.Our findings indicated that hypoxia could increase pulmonary arterial pressure, wall thickness ratio of pulmonary artery, and right ventricular hypertrophy as well as downregulate eNOS, sGCα1 and PKG protein expression whereas upregulate ROCK II and VEGF protein expression in molecular mechanism. However, the above effects could be reversed by treating with KMUP-1 or Sildenafil. In conclusion, our study confirmed that KMUP-1 is involved in the expression of eNOS/sGCα1/PKG signaling pathway resulting in vessel relaxation and may be useful for the improvement of hypoxia-induced pulmonary arterial hypertension in the future.

Thesis (M.Sc.)--York University, 2005. Graduate Programme in Biology.
Typescript. Includes bibliographical references (leaves 61-76). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11893

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin.
Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells.
The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation.
The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis.
The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets.
Leung Lai-han.
"July 2006."
Adviser: Ronald Ray Fiscus.
Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (p. 175-191).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Blutplättchen spielen unter physiologischen Bedingungen eine wichtige Rolle bei der Erhaltung der Hämostase. So verhindern sie ein andauerndes Bluten von Wunden, indem sie in Blutgefässen zwischen normalen Zellen des Endothels und beschädigten Bereichen unterscheiden und sich dort gezielt anheften können. Das Zusammenspiel der Plättchenagonisten und den dazugehörigen Rezeptoren wird durch intrazelluläre Signalmoleküle kontrolliert, die die Aktivierung der Blutplättchen regulieren. Äusserst wichtige intrazellulare Signalmoleküle stellen dabei die zyklischen Nukleotide cGMP und cAMP dar, die bei der Hemmung der Plättchen beteiligt sind. Die Bildung von cGMP und cAMP in den Blutplättchen wird durch die aus dem Endothel freigesetzten Moleküle NO und Prostacyclin (PGI2) stimuliert, die ihrerseits Blutplättchen hemmen, indem sie Proteinkinase G (PKG) und Proteinkinase A (PKA) aktivieren. Neuerdings wird vorgeschlagen, dass es sich bei ROS („reactive oxygen species“) um einen neuen Modulator bei der Signaltransduktion zwischen verschiedenen Zelltypen handelt. Die hier zusammengefasste Arbeit beschreibt die Rolle der ROS-Produktion bei der Aktivierung von Blutplättchen, die Beziehung zwischen dem NO/cGMP/PKG I Signalweg und der ROS bzw. MAP-Kinase Signaltransduktion, und die Rolle von zyklischen Nukleotiden bei der Entwicklung von Megakaryozyten und Blutplättchen. Werden Blutplättchen durch unterschiedliche Einflüsse aktiviert, so produzieren sie über die Aktivierung von NAD(P)H-Oxidase nur intrazelluläres aber nicht extrazelluläres ROS. Dabei beinflusst das in den Blutplättchen produzierte ROS signifikant die Aktivierung von αIIbβ3 Integrin, nicht jedoch die Sekretion von alpha- bzw. dichten Granula oder die Gestalt der Blutplättchen. Die Thrombin-induzierte Integrin αIIbβ3-Aktivierung ist nach Behandlung der Blutplättchen mit Hemmstoffen der NAD(P)H-Oxidase oder Superoxid-Fängern signifikant reduziert. Diese Inhibitoren reduzieren auch die Aggregation der Blutplättchen bzw. die Thrombusbildung auf Kollagen, wobei diese Effekte unabhängig vom NO/cGMP Signalweg vermittelt werden. Sowohl ADP, das von dichten Granula der Blutplättchen sezerniert wird und zur Aktivierung von P2Y12-Rezeptoren führt, als auch die Freigabe von Thromboxan A2 stellen wichtige, vorgeschaltete Vermittler bei der p38 MAP Kinase-Aktivierung durch Thrombin dar. Jedoch spielt die p38 MAP-Kinase-Aktivierung keine signifikante Rolle bei der Thrombin-induzierten Kalzium-Mobilisierung, P-Selektin Exprimierung, αIIbβ3 Integrin Aktivierung oder Aggregation der Blutplättchen. Abschliessend kann festgestellt werden, dass sich die Aktivierung der PKG insgesamt klar hemmend auf die p38 and ERK MAP-Kinasen in menschlichen Blutplättchen auswirkt. Desweiteren zeigt diese Studie, dass zyklische Nukleotide nicht nur die Blutplättchen hemmen, sondern auch einen Einfluss auf die Entwicklung der Megakaryozyten und Blutplättchen haben, aber auf unterschiedliche Weise. cAMP ist an der Differenzierung von embryonalen hämatopoietischen Zellen zu Megakaryozyten beteiligt, wobei cGMP keine Rolle bei diesem Prozess spielt. Während PKA in embryonalen Zellen schon vertreten ist, steigt beim Reifungsprozess der Megakaryozyten die Expression von Proteinen, die bei der cGMP Signalverbreitung („soluble guanylyl cyclase“, sGC; PKG) mitwirken, stetig an. In der letzten Phase der Reifung von Megakaryozyten, die durch die Freisetzung der Blutplättchen charakterisiert ist, zeigen cGMP und cAMP leicht divergierende Effekte: cGMP verstärkt die Bildung von Blutplättchen, während cAMP dieselbe reduziert. Dies deutet auf einen fein abgestimmten Prozess hin, abhängig von einem Stimulus, der von den benachbarten Zellen des Sinusoid-Endothels stammen könnte. Die Ergebnisse dieser Dissertation tragen zu einen besseren Verständnis der Regulation von Blutplättchen sowie der möglichen molekularen Mechanismen bei, die eine Rolle bei der Reifung von Megakaryozyten im vaskularen Mikroumfeld des Knochenmarks innehaben
In physiological conditions platelets have a major role in maintaining haemostasis. Platelets prevent bleeding from wounds by distinguishing normal endothelial cells in vasculature from areas with lesions to which they adhere. Interaction of platelet agonists and their receptors is controlled by intracellular signaling molecules that regulate the activation state of platelets. Very important intracellular signaling molecules are cyclic nucleotides (cGMP and cAMP), both involved in inhibition of platelet activation. Formation of cGMP and cAMP in platelets is stimulated by endothelial-derived NO and prostacyclin (PGI2), which then mediate inhibition of platelets by activating protein kinase G (PKG) and protein kinase A (PKA). Recently, it has been suggested that reactive oxygen species (ROS) represent new modulators of cell signaling within different cell types. The work summarized here describes the involvement of platelet ROS production in platelet activation, the relation of NO/cGMP/PKG I pathway to ROS and to mitogen-activated protein kinases (MAP kinase) signaling, and the involvement of cyclic nucleotides in megakaryocyte and platelet development. Platelets activated with different agonists produce intracellular but not extracellular ROS by activation of NAD(P)H oxidase. In addition, ROS produced in platelets significantly affects αIIbβ3 integrin activation but not alpha/dense granule secretion and platelet shape change. Thrombin induced integrin αIIbβ3 activation is significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors and superoxide scavengers. These inhibitors also reduce platelet aggregation and thrombus formation on collagen under high shear and achieve their effects independently of the NO/cGMP pathway. ADP secreted from platelet dense granules with subsequent activation of P2Y12 receptors as well as thromboxane A2 release are found to be important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation does not significantly contribute to calcium mobilization, P-selectin expression, αIIbβ3 integrin activation and aggregation of human platelets in response to thrombin. Finally, PKG activation does not stimulate, but rather inhibit, p38 and ERK MAP kinases in human platelets. Further study revealed that cyclic nucleotides not only inhibit platelet activation, but are also involved, albeit differentially, in megakaryocyte and platelet development. cAMP is engaged in haematopoietic stem cell differentiation to megakaryocytes, and cGMP has no impact on this process. While PKA is already present in stem cells, expression of proteins involved in cGMP signaling (soluble guanylyl cyclase, sGC; PKG) increases with maturation of megakaryocytes. In the final step of megakaryocyte maturation that includes release of platelets, cGMP and cAMP have mild but opposing effects: cGMP increases platelet production while cAMP decreases it indicating a finely regulated process that could depend on stimulus coming from adjacent endothelial cells of sinusoids in bone marrow. The results of this thesis contribute to a better understanding of platelet regulation and of the possible molecular mechanisms involved in megakaryocyte maturation in bone marrow vascular microenvironment