No / We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Guanilina (GN), uroguanilina (UGN) e a enterotoxina termo-estÃvel da Escherichia coli (STa) fazem parte de uma nova famÃlia de peptÃdeos que ativam a formaÃÃo de cGMP. A ingestÃo de sal na dieta induz a secreÃÃo de GN e UGN no lÃmen intestinal, inibindo a reabsorÃÃo de sÃdio e induzindo a secreÃÃo de Cl-, HCO3- e Ãgua. Simultaneamente, esses hormÃnios estimulam a excreÃÃo renal de eletrÃlitos atravÃs da induÃÃo de natriurese, caliurese e diurese. Esse mecanismo altamente integrado permite a manutenÃÃo do sÃdio corporal, atravÃs da eliminaÃÃo desse excesso de sÃdio pela urina. Entretanto, essa regulaÃÃo fisiolÃgica entre o intestino e o rim na tem sido muito bem estudada. O objetivo desse trabalho à estudar as alteraÃÃes no metabolismo diÃrio e na funÃÃo renal de ratos submetidos a uma alta ingestÃo de cloreto de sÃdio. Os efeitos forma examinados usando ratos Wistar mantidos por 10 dias em gaiolas metabÃlicas. O grupo controle recebeu somente Ãgua destilada, enquanto que os grupos tratados receberam 1% e 2% de soluÃÃo de NaCl. Forma analisadas diariamente o volume urinÃrio, o peso corporal e o consumo de Ãgua e alimento. A funÃÃo renal foi avaliada atravÃs da perfusÃo de rim isolado de ratos apÃs dez dias de tratamento em gaiolas metabÃlicas, onde a perfusÃo foi realizada com soluÃÃo de Krebs-Henseleit modificada com 6g% de albumina bovina. Os dados foram comparados atravÃs de teste t de Student e ANOVA, com significÃncia p<0,05. O peso dos ratos tratados com 2% de NaCl apresentou uma reduÃÃo a partir do dia 8, em relaÃÃo ao controle, enquanto que 1% nÃo apresentou reduÃÃo significativa. O volume urinÃrio e o consumo de Ãgua apresentaram um aumento em ambos os tratamentos a partir do dia 2. O consumo de alimento ingerido nÃo apresentou grandes variaÃÃes entre os grupos. Em rim isolado de rato ambos os tratamentos aumentou a pressÃo de perfusÃo (PP). A resistÃncia vascular renal (RVR), o fluxo urinÃrio (FU), o ritmo de filtraÃÃo glomerular (RFG) e o clearance osmolar (Cosm) aumentou no grupo de 1% comparado ao controle, porÃm esses mesmos parÃmetros diminuÃram no grupo de 2%. Em relaÃÃo ao transporte de eletrÃlitos observa-se alteraÃÃo somente no grupo de 2%, onde reduziu o transporte de sÃdio (%TNa+, %pTNa+), potÃssio (%TK+, %pTK+) e cloreto (%TCl-, %pTCl-). Esses resultados sugerem que a alta ingestÃo de NaCl na dieta promove significativa alteraÃÃes no metabolismo diÃrio e na funÃÃo renal. / Guanylin (GN), uroguanylin (UGN), and the bacterial heat-stable enterotoxin (ST) peptides comprise a new family of cyclic guanosine 3â-5â monophosphate (cGMP)-regulating agonist. Ingestion of a salt meal induces secretion of GN and UGN into the intestinal lumen, where they inhibit Na+ absorption and induces Cl-, HCO3- , and water secretion. Simultaneously, these hormones stimulate renal electrolyte excretion by inducing natriuresis, kaliuresis, and diuresis. The highly integrated mechanism allows the organism to maintain sodium balance by eliminating excess NaCl in the urine. However, their physiological regulation within the kidney has not been studied. The aim of this study was showing changes on daily metabolism and renal function of rat under high sodium chloride ingestion. Its effects were examined using wistar rats maintained for ten days in metabolic cages. Control group received only water, two more groups received 1% and 2% solutions of sodium chlroride. We daily analyzed urinary volume, weigh, and food and water consume. The renal function was evaluated using isolated perfused kidneys, in which the kidneys were perfused after ten days in metabolic cages, only with Krebs-Henseleit solution containing 6g% of a previously dialysed bovine albumine serum. All data were analyzed by ANOVA and Student t-test with level of significance set at *p<0,05. Ratâs weights of 2% group decreased after eighth day, compared with control group, while 1% group did not show significative weight lost. Urinary volume and water consume increased, in both treatments, from second day. Food consume did not show significative among groups. In isolated kidney both treatments increase perfusion pressure (PP). The renal vascular resistence (RVR), urinary flow (UF), glomerular filtration rate (GFR) and the osmolar clearance (Cosm) increased in the 1% group compared with control group, however decreased in 2% NaCl group. Treatment with 2% NaCl decreased the sodium (%TNa+, %pTNa+), potassium (%TK+, %pTK+) and chloride (%TCl-, %pTCl-). These results suggest that a high salt ingestion on diet promote significative changes on daily metabolism and the renal function of rats.

Introduction: 23 million patients worldwide suffer from heart failure. These patients depend on cardiac research, because cardiac research enables the development of new therapeutic strategies and –targets. In cardiomyocytes, the compartmentalization of cAMP and cGMP depends on many factors. T-tubuli and PDEs are responsible for the division of cells in microdomains in which localized and specific cAMP and cGMP-signaling occurs. The aim of this thesis was to develop a method to answer the open questions that remain about the physiological and pathophysiological significance of cAMP/cGMP compartmentalization. Methods: I used the zebrafish as a model, because the transparency of zebrafish larvae enabled non-invasive fluorescent imaging in cardiomyocytes in the living animal. I cloned the Fluorescence Resonance Energy Transfer (FRET) sensors EPAC1-camps for cAMP and cGi500 for cGMP and injected them into zebrafish fertilized embryos. Then I used the F0 generation for Fluorescence Lifetime Imaging (FLIM) -FRET-measurements of cAMP and cGMP. Ca2+ is an important downstream mediator of cAMP and cGMP, because Ca2+ regulates cardiac contraction. Therefore, I also cloned the Ca2+ sensor GCaMP6 and used the dye Fluo-4 AM to include intracellular Ca2+ in the imaging. Results: The cloned sensors for cAMP, cGMP and Ca2+ were successfully injected into the zebrafish and showed expression in individual cardiomyocytes. I developed a protocol to mount the living zebrafish embryos and to measure intracellular cAMP and cGMP with FLIM-FRET in vivo with high spatial resolution. I characterized the sensors in their functionality by showing that the sensors react to changes in intracellular concentrations of cAMP and cGMP. The results of this study include evidence that zebrafish have mechanisms that lead to cAMP/cGMP compartmentalization in the absence of T-tubuli, and these mechanisms keep compartmentalization constant even under extreme cAMP or cGMP increasing drug treatment. Furthermore, I imaged intracellular Ca2+ by confocal microscopy and developed a protocol to use Fluo-4 AM for Ca2+ imaging. Conclusion: The method used in this thesis should allow the investigation of subcellular cAMP/cGMP compartmentalization and Ca2+ and to subsequently answer open questions in the field, for example whether a change of cAMP compartmentalization leads to the pathological phenotypes of cardiac disease or if a changed compartmentalization of cAMP in cardiac disease influences Ca2+ concentrations and therefore contraction. Additionally, this method can be used to learn more about cAMP, cGMP und Ca2+ during regeneration in the heart, because the zebrafish cardiomyocytes can regenerate. / Einleitung: Weltweit sind mehr als 23 Millionen unter Herzinsuffizienz leidende Patienten auf die kardiologische Grundlagenforschung angewiesen, da diese die Voraussetzung für eine bessere Versorgung durch adaptierte und neue Behandlungswege schafft. In Kardiomyozyten hängt die Kompartimentierung von cAMP und cGMP von vielen Faktoren ab. T-Tubuli und PDEs werden unter anderem für die Aufteilung der Zellen in Mikrodomänen, in denen lokalisierte und spezifische cAMP- und cGMP-Signalgebung stattfinden kann, verantwortlich gemacht. Das Ziel dieser Arbeit war die Etablierung einer Methode, mithilfe derer offene Fragen bezüglich der physiologischen und insbesondere der pathophysiologischen Relevanz der cAMP- und cGMP Kompartimentierung beantwortet werden können. Methode: Als Modell diente der Zebrafisch, da die Transparenz von Zebrafisch Embryonen eine nicht-invasive Bildgebung von Fluoreszenz in Kardiomyozyten im lebenden Tier ermöglicht. Dafür klonierte ich die Förster Resonance Energy Transfer (FRET) -Sensoren EPAC1-camps als cAMP-Sensor und cGi500 als cGMP-Sensor und injizierte diese in befruchtete Zebrafisch Embryonen. Anschließend benutzte ich die F0-Generation für Fluorescence Lifetime Imaging (FLIM) -FRET-Messungen von cAMP und cGMP. Da Ca2+ als wichtiger downstream Mediator von cAMP und cGMP die kardiale Kontraktion reguliert, klonierte ich außerdem den Ca2+-Sensor GCaMP6 und benutzte den Farbstoff Fluo-4 AM, um intrazelluläres Ca2+ darzustellen. Ergebnisse: Die klonierten Sensoren für cAMP, cGMP und Ca2+ konnten erfolgreich in den Zebrafisch injiziert werden und zeigten alle Expression in einzelnen Kardiomyozyten. Ich entwickelte ein Protokoll, dass die Fixierung von lebenden Zebrafisch Embryonen und nachfolgender Bildgebung von cAMP und cGMP mit hoher zellulärer Auflösung mit FLIM-FRET in vivo erlaubte. Ich konnte eine funktionelle Charakterisierung der Sensoren durchführen, indem ich zeigte, dass sie auf Konzentrationsänderungen von intrazellulärem cAMP und cGMP reagieren sowie zeigen, dass Zebrafische trotz fehlender T-Tubuli eine signifikante cAMP- und cGMP Kompartimentierung aufweisen, auch unter extremen Bedingungen nach Gabe von cAMP/cGMP stimulierenden Substanzen in hoher Dosierung. Ich konnte zudem subzelluläres Ca2+ durch konfokale Mikroskopie bildgebend darstellen und entwickelte ein Protokoll, um mit Fluo-4 AM eine schnelle Möglichkeit zu haben, Ca2+ mit in die Messungen einzubeziehen. Ausblick: Die in dieser Arbeit benutzte Methode bietet eine gute Möglichkeit, subzelluläre cAMP- und cGMP-Kompartimentierung und Ca2+ zu untersuchen und damit zum Beispiel die Fragen zu beantworten, ob eine veränderte cAMP/cGMP Kompartimentierung zu Herzkrankheiten wie Hypertrophie führt oder ob eine veränderte cAMP Kompartimentierung den zellulären Ca2+ Haushalt und damit die kardiale Kontraktion beeinflusst. Darüber hinaus kann das von mir etablierte Protokoll dazu genutzt werden, mehr über cAMP, cGMP und Ca2+ während der Regeneration im Herzen zu lernen, da der Zebrafisch über ausgeprägte Regenerationsfähigkeiten verfügt.

Introduction: 23 million patients worldwide suffer from heart failure. These patients depend on cardiac research, because cardiac research enables the development of new therapeutic strategies and –targets. In cardiomyocytes, the compartmentalization of cAMP and cGMP depends on many factors. T-tubuli and PDEs are responsible for the division of cells in microdomains in which localized and specific cAMP and cGMP-signaling occurs. The aim of this thesis was to develop a method to answer the open questions that remain about the physiological and pathophysiological significance of cAMP/cGMP compartmentalization. Methods: I used the zebrafish as a model, because the transparency of zebrafish larvae enabled non-invasive fluorescent imaging in cardiomyocytes in the living animal. I cloned the Fluorescence Resonance Energy Transfer (FRET) sensors EPAC1-camps for cAMP and cGi500 for cGMP and injected them into zebrafish fertilized embryos. Then I used the F0 generation for Fluorescence Lifetime Imaging (FLIM) -FRET-measurements of cAMP and cGMP. Ca2+ is an important downstream mediator of cAMP and cGMP, because Ca2+ regulates cardiac contraction. Therefore, I also cloned the Ca2+ sensor GCaMP6 and used the dye Fluo-4 AM to include intracellular Ca2+ in the imaging. Results: The cloned sensors for cAMP, cGMP and Ca2+ were successfully injected into the zebrafish and showed expression in individual cardiomyocytes. I developed a protocol to mount the living zebrafish embryos and to measure intracellular cAMP and cGMP with FLIM-FRET in vivo with high spatial resolution. I characterized the sensors in their functionality by showing that the sensors react to changes in intracellular concentrations of cAMP and cGMP. The results of this study include evidence that zebrafish have mechanisms that lead to cAMP/cGMP compartmentalization in the absence of T-tubuli, and these mechanisms keep compartmentalization constant even under extreme cAMP or cGMP increasing drug treatment. Furthermore, I imaged intracellular Ca2+ by confocal microscopy and developed a protocol to use Fluo-4 AM for Ca2+ imaging. Conclusion: The method used in this thesis should allow the investigation of subcellular cAMP/cGMP compartmentalization and Ca2+ and to subsequently answer open questions in the field, for example whether a change of cAMP compartmentalization leads to the pathological phenotypes of cardiac disease or if a changed compartmentalization of cAMP in cardiac disease influences Ca2+ concentrations and therefore contraction. Additionally, this method can be used to learn more about cAMP, cGMP und Ca2+ during regeneration in the heart, because the zebrafish cardiomyocytes can regenerate. / Einleitung: Weltweit sind mehr als 23 Millionen unter Herzinsuffizienz leidende Patienten auf die kardiologische Grundlagenforschung angewiesen, da diese die Voraussetzung für eine bessere Versorgung durch adaptierte und neue Behandlungswege schafft. In Kardiomyozyten hängt die Kompartimentierung von cAMP und cGMP von vielen Faktoren ab. T-Tubuli und PDEs werden unter anderem für die Aufteilung der Zellen in Mikrodomänen, in denen lokalisierte und spezifische cAMP- und cGMP-Signalgebung stattfinden kann, verantwortlich gemacht. Das Ziel dieser Arbeit war die Etablierung einer Methode, mithilfe derer offene Fragen bezüglich der physiologischen und insbesondere der pathophysiologischen Relevanz der cAMP- und cGMP Kompartimentierung beantwortet werden können. Methode: Als Modell diente der Zebrafisch, da die Transparenz von Zebrafisch Embryonen eine nicht-invasive Bildgebung von Fluoreszenz in Kardiomyozyten im lebenden Tier ermöglicht. Dafür klonierte ich die Förster Resonance Energy Transfer (FRET) -Sensoren EPAC1-camps als cAMP-Sensor und cGi500 als cGMP-Sensor und injizierte diese in befruchtete Zebrafisch Embryonen. Anschließend benutzte ich die F0-Generation für Fluorescence Lifetime Imaging (FLIM) -FRET-Messungen von cAMP und cGMP. Da Ca2+ als wichtiger downstream Mediator von cAMP und cGMP die kardiale Kontraktion reguliert, klonierte ich außerdem den Ca2+-Sensor GCaMP6 und benutzte den Farbstoff Fluo-4 AM, um intrazelluläres Ca2+ darzustellen. Ergebnisse: Die klonierten Sensoren für cAMP, cGMP und Ca2+ konnten erfolgreich in den Zebrafisch injiziert werden und zeigten alle Expression in einzelnen Kardiomyozyten. Ich entwickelte ein Protokoll, dass die Fixierung von lebenden Zebrafisch Embryonen und nachfolgender Bildgebung von cAMP und cGMP mit hoher zellulärer Auflösung mit FLIM-FRET in vivo erlaubte. Ich konnte eine funktionelle Charakterisierung der Sensoren durchführen, indem ich zeigte, dass sie auf Konzentrationsänderungen von intrazellulärem cAMP und cGMP reagieren sowie zeigen, dass Zebrafische trotz fehlender T-Tubuli eine signifikante cAMP- und cGMP Kompartimentierung aufweisen, auch unter extremen Bedingungen nach Gabe von cAMP/cGMP stimulierenden Substanzen in hoher Dosierung. Ich konnte zudem subzelluläres Ca2+ durch konfokale Mikroskopie bildgebend darstellen und entwickelte ein Protokoll, um mit Fluo-4 AM eine schnelle Möglichkeit zu haben, Ca2+ mit in die Messungen einzubeziehen. Ausblick: Die in dieser Arbeit benutzte Methode bietet eine gute Möglichkeit, subzelluläre cAMP- und cGMP-Kompartimentierung und Ca2+ zu untersuchen und damit zum Beispiel die Fragen zu beantworten, ob eine veränderte cAMP/cGMP Kompartimentierung zu Herzkrankheiten wie Hypertrophie führt oder ob eine veränderte cAMP Kompartimentierung den zellulären Ca2+ Haushalt und damit die kardiale Kontraktion beeinflusst. Darüber hinaus kann das von mir etablierte Protokoll dazu genutzt werden, mehr über cAMP, cGMP und Ca2+ während der Regeneration im Herzen zu lernen, da der Zebrafisch über ausgeprägte Regenerationsfähigkeiten verfügt.

Die Azidose gehört zu den stärksten dilatatorischen Stimuli zerebraler Arterien. Obwohl schon 1890 von Roy und Sherrington beschrieben, sind die Faktoren, die die Vasodilatation zerebraler Arterien auf Azidose vermitteln, bis heute nicht bekannt. Untersuchungen über die Rolle des schnell flüchtigen Bioradikals Stickstoffmonoxid (NO) haben gezeigt, daß NO bei der azidotischen Vasodilatation zerebraler Arterien als Modulator agiert. Darüber hinaus nimmt NO in der neurovaskulären Kopplung, d.h. bei der Vermittlung der regionalen Blutflußantwort nach neuronaler Stimulation, eine permissive Funktion ein. Die Vasodilatation auf Azidose wurde in der vorliegenden Arbeit als Modellstimulus zur Untersuchung der NO-abhängigen Dilatation zerebraler Arterien verwendet. Dabei wurde die Rolle der Kaliumkanäle und die Funktion des cGMP an der Vasodilatation auf Azidose mittels spezifischer Inhibitoren untersucht. Die Experimente erfolgten an der isolierten und perfundierten A. cerebri media der Ratte. Bei der Untersuchung der Signaltransduktion von NO auf Ebene des cGMP wurde eine ausgeprägte Abhängigkeit der azidotischen Vasodilatation von cGMP beobachtet. Durch Restitution des basalen cGMP-Spiegels nach vorheriger Inhibition der löslichen Guanylatzyklase wurde gezeigt, daß NO über cGMP bei der Vermittlung dieser Reaktion als Modulator wirkt. Unter Blockade der einzelnen Kaliumkanalfamilien konnte eine Beteiligung der KCa an der Vasodilatation auf Azidose sowie am Gefäßtonus unter Ruhebedingungen beobachtet werden. Für eine Beteiligung der KATP, der KV und der Kir an diesen Reaktionen wurden dagegen keine Hinweise gefunden. Ebenso sprechen die Untersuchungen unter Blockade der Na+/K+-ATPase gegen eine Beteiligung dieses Enzyms an der Azidosereaktivität zerebraler Arterien. Um ein mögliches Zusammenwirken der Kaliumkanäle zu erfassen, wurde die Vasodilatation auf Azidose unter Blockade von jeweils zwei Kaliumkanaltypen untersucht. Unter Hemmung der KCa und der KATP, sowie unter Hemmung der BKCa und der KATP wurde keine Vasodilatation mehr auf Azidose beobachtet. Die Ergebnisse sprechen dafür, daß die Vasodilatation der A. cerebri media auf Azidose durch BKCa und KATP in redundanter Weise vermittelt wird. Dabei scheinen KCa die Funktion der KATP vollständig substituieren zu können. Die Resultate dieser Arbeit bilden den Ausgangspunkt für derzeit laufenden Untersuchungen über die funktionelle Modulation der KATP und der BKCa durch das NO/cGMP-System. Weiterhin bilden die vorliegenden Untersuchungen eine wichtige Grundlage zur Überprüfung der zentralen Rolle der KCa und der KATP auf weitere, durch das NO/cGMP-System modulierten Stimuli, wie z.B. der funktionellen Stimulation. Die in dieser Arbeit vorgestellten Experimente wurden mit Mitteln der Deutschen Forschungsgemeinschaft (SFB 507), der Hermann und Lilly Schilling Stiftung, sowie der Humboldt Universität zu Berlin gefördert. / Acidosis is one of the most potent vasodilators in the cerebral circulation. Although first described 1890 by Roy and Sherrington the mechanisms of vasodilation to acidosis are still unknown. Experimental data show, that nitric oxide (NO) is a modulator but not a mediator of cerebral arterial pH reactivity. NO also acts as a modulator of neurovascular coupling in the rat somatosensory cortex. We used the experimental in vitro model of the isolated and perfused middle cerebral artery (MCA) to elucidate the general mechanisms of NO-modulated dilations. The present study was performed to clarify the role of cGMP and potassium channels for mediation of acidosis-induced dilation of cerebral arteries. The results indicate, that vasodilation to acidosis is mediated by cGMP. Restoring the basal cGMP-level we could demonstrate a permissive role of cGMP in the vasodilation to acidosis. We could also show that KCa are active under resting conditions and are able to contribute to the relaxation of the MCA to acidosis. Other potassium channels like KATP, Kir, KV and the Na+/K+ATPase appeared not to be involved in the process of dilation to acidosis. After administration of a selective inhibitor of KATP in addition to an inhibitor of KCa the relaxation to acidosis was completely abolished. Simultaneous application of selective inhibitors of KATP and BKCa also prevented from vasodilation to acidosis. These results indicate, that relaxation to acidosis is mediated by activation of KATP and BKCa. This potassium channels seem to have a redundant activity, in such a way that KCa could substitute for KATP. The present findings are a starting point for further studies concerning the modulation of KATP and BKCa by the NO/cGMP-System. This studies are a basis for coming experiments to determine the role of KATP and BKCa in the neurovascular coupling.

Soluble guanylyl/guanylate cyclase (sGC), the primary receptor for nitric oxide (NO), is a heme containing heterodimeric enzyme involved in numerous physiological events in animals. The small molecule YC-1 stimulates sGC, but the mechanism behind and the location of binding are unknown. I have developed a prokaryotic expression system for insect ( <italic>Manduca sexta</italic>) sGC. The recombinant holoenzyme, like its mammalian counterpart, is responsive to NO, CO and YC-1, displaying a 175-fold increase in activity on binding. Truncated constructs containing the N-terminal two-thirds of both subunits (msGC-NT) were designed to facilitate expression. With the highly pure material, we investigated NO and CO binding, reaction kinetics and regulation. Binding of NO to msGC-NT heme forms a six-coordinate intermediate followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex. The conversion rate is insensitive to nucleotides, YC-1 and changes in NO concentration up to ~30 micromolar. In contrast, NO release from msGC-NT is biphasic in the absence of YC-1, while binding of YC-1 eliminates the fast phase but has little effect on the slower phase. CO binding to msGC-NT is also regulated by YC-1. The CO release rate is reduced by YC-1 while the on rate remains unchanged, which leads to an ~50-fold increase in binding affinity. YC-1 binding leads to a substantial geminate recombination of CO to msGC-NT upon photolysis. Our data are consistent with a model for allosteric activation in which (1) YC-1 binds away from the catalytic site and (2) sGC undergoes a conformational switch between two states of an open and a closed heme pocket. The final catalysis results from the integration of the influence of numerous allosteric effectors on the equilibrium between these two states.<italic>S </italic>-nitrosoglutathione (GSNO) exists <italic>in vivo </italic> and plays important roles in NO signaling. We have developed a model cell line, in which inducible NO synthase and human sGC genes were included. GSNO stimulation of sGC has been investigated using recombinant insect and human enzymes. GSNO can activate sGC as efficiently as gaseous NO, but apparently with a distinct mechanism. GSNO or endogenous NO could <italic>S </italic>-nitrosylate sGC, which might regulate the enzyme function.

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.

The worm Caenorhabditis elegans is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today. In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGFβ ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.

The role of cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG) in necrotic and apoptotic pathways of many cell types is well established; however its role in the ischemic preconditioning (IPC) of cardiomyocytes is not clearly defined. In the current study, we assessed the hypothesis that PKG protects against cell death following ischemidreperfusion injury in myocytes subjected to IPC. Freshly isolated adult rat ventricular myocytes were subjected to IPC by incubating in ischemic buffer for 30 minutes (min) followed by incubation in normal medium for 30 min. Prolonged simulated ischemia (SI) was created by incubating myocytes in the ischemic buffer for 90 min and reoxygenation (RO) for 120 min in the normal medium. Necrosis was determined by trypan blue exclusion and apoptosis was assessed by TUNEL assay. IPC reduced necrosis as shown by significant decrease in trypan blue positive cells as compared to virgin non-preconditioned myocytes subjected to SI and RO alone (p<.01). Similarly, the number of TUNEL positive myocytes following SI and 18 hrs of RO were significantly reduced in the IPC group. Treatment with PKG inhibitor, KT5832 (2pM) completely abolished the protection against necrosis by IPC. However, KT5832 failed to abolish the protective affect of IPC against apoptosis. Furthermore, myocytes infected with an adenoviral construct of PKG-la (1 x 1 o4 particles/cell) significantly reduced the number of trypan blue and TUNEL positive cells. These results suggest that the PKG signaling pathway plays an essential role in the preconditioning of myocytes against necrosis following SI / RO injury. Furthermore, while the overexpression of PKG protects myocytes against necrosis, as well as apoptosis, IPC may not induce a sufficient level of PKG during 18 hours of RO to induce protection against apoptosis.

O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos. / The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.