@phdthesis{Makiol2014, author = {Makiol, Christian}, title = {Die Rolle der durch NADPH-Oxidasen produzierten reaktiven Sauerstoffspezies (ROS) in der Pathophysiologie von Hypertonie und Alterung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109901}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Reaktive Sauerstoffradikale spielen eine große Rolle bei der Entstehung von Karzinomen, im Alterungsprozess und bei kardiovaskul{\"a}ren Erkrankungen (Valko et al., 2007). Solche Sauerstoffradikale k{\"o}nnen unter anderem durch die NADPH-Oxidase gebildet werden (Finkel and Holbrook, 2000). Ziel der Arbeit war es, die Rolle von ROS im Alterungsprozess und bei Bluthochdruck aufzuzeigen. Daf{\"u}r wurden zwei Tiermodelle, eines mit einer geringen ROS-Konzentration und eines mit einer hohen ROS-Konzentration, verwendet. Zum einen handelt es sich um ein p47-Knockout-Mausmodell f{\"u}r die geringere ROS-Konzentration, im Vergleich zu alten und jungen Wildtyp-Tieren. F{\"u}r die Rolle von ROS bei der Alterung wurden junge mit alten Wildtyp-Tieren verglichen. Um die Rolle der NOX2 in den ROS-Spiegeln der Organe zu ermitteln wurden gleichaltrige Wildtyp- mit p47-Knockout-Tieren verglichen. Zum anderen nutzten wir ein ren2-Tiermodell f{\"u}r die hohe ROS-Konzentration. Hierbei handelt es sich um ein Bluthochdruckmodell, in dem der Blutdruck mit Medikamenten normalisiert werden kann. Die Tiere wurden daher mit Ramipril (ACE-Inhibitor), Apocynin (NADPH-Oxidase-Inhibitor) und Tempol (Radikalf{\"a}nger) behandelt. Als Maß f{\"u}r die ROS-Konzentration wurden die Superoxidionenspiegel mithilfe einer Dihydroethidium-F{\"a}rbung ermittelt. Die DNA-Doppelstrangbr{\"u}che wurden mit einer γH2AX-Antik{\"o}rper-F{\"a}rbung nachgewiesen und die Expression von Sirtuin1 und Hsp70, welche als Anti-Aging Proteine bekannt sind, mittels Western Blot bestimmt.}, subject = {Reaktive Sauerstoffspezies}, language = {de} } @phdthesis{Mandel2014, author = {Mandel, Philipp}, title = {Entstehung von oxidativen Stressmarkern in DNA und RNA nach der Behandlung mit den Hormonen Angiotensin II und Aldosteron in vitro und in vivo : Vergleich von drei Analysemethoden zum Nachweis von 8-Oxo-2'-desoxyguanosin in LLC-PK1-Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111190}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The detection of oxidative stress markers has gained increasing importancy in the early investigation of diseases like diabetes, cancer or hypertension. 8 oxo 2' deoxyguanosine (8-oxodG) is the main marker, which is used for the intracellular detection of oxidative stress levels. However, the oxidative stress markers 8 oxoguanine (8-oxoGua), a product of the DNA base excision repair and 8 oxoguanosine (8-oxoGuo), a marker for oxidative damaged RNA have received less attention up to now. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation processes of the blood pressure system. During hypertension angiotensin II (Ang II) and aldosterone (Aldo) are released in high concentrations over a longer period leading to non-physiological effects of the RAAS hormones. Subsequently, an increase of the intracellular oxidative stress level in kidney cells can be measured. The aim of this thesis is the in vitro and in vivo detection of the oxidative damage in DNA and RNA by measuring oxidative stress markers, especially 8-oxodG which is triggered by Ang II and Aldo. In vitro experiments were carried out in LLC-PK1, a cell line originated from porcine kidney cells. It could been shown that Ang II and Aldo led to a dose-dependent increase of DNA damage in the cells. A time-dependent increase was detected for the first 30 minutes of the treatment. For the rest of the experimental set up (4 h) the level of detected DNA damage remained constant. The FPG comet assay and the immunocytochemical staining showed a significant increase of 8-oxodG in the cells, whereas the HPLC-MS/MS measurement only detected a small increase of 8-oxodG in the DNA. The FPG enzyme, which recognises also other oxidized purines besides 8-oxodG, which led to an overestimation of 8-oxodG in the comet assay. Also, the 8 oxodG antibody, which was used in the immunocytochemical analysis, detected higher amounts of 8-oxodG most likely due to its side reactions with other oxidized DNA structures. One of the main advantages of the last mentioned methods is the direct measurement in damaged cells, whereas the HPLC-MS/MS requires an isolation of the DNA. During this isolation process the oxidative stress markers can be oxidized and the detection can become imprecise. The main purpose of the in vivo experiments was the detection of the oxidative stress marker 8-oxoGua, 8-oxodG and 8-oxoGuo in the urine of test animals. The treatment of C57BL/6 mice and Sprague Dawley (SD) rats with the RAAS hormones led to an increase of the blood pressure, higher DNA damage due to oxidative stress as well as an increased excretion rate of oxidative stress markers. The inhibition of the angiotensin II type 1- or mineralocorticoid receptor and a mutation of the AT1a gene could show, that the DNA damage is independent from the hypertension. In addition, it was shown that the NOX4 is not alone responsible for the oxidative stress. Other NADPH oxidases must contribute to the induction of oxidative stress inside the cell. Moreover, the activation of the Nrf2 pathway has an influence on the effect of Aldo in SD rats. The excretion rate of the oxidative stress markers in the 20 h urine of the treated animals showed how the equilibrium between the DNA repair and the oxidative stress level was changing over time. The measurement of 8-oxoGuo became more and more popular, because up to the fact that 80 \% of the DNA is translated into RNA. Overall, the detection of 8-oxodG and 8-oxoGuo is feasible for monitoring the disease or the healing process, because the measurement is non-invasive. The detection of 8-oxodG and 8-oxoGuo in nucleic acids is a first step into the field of basic research methods, because it reveals a snapshot of the nucleic acid damage in the cell at a specific time point. Usually, there will be an overestimation of the oxidative stress marker resulting from the analytical method. Although, it is possible to detect an underestimation of oxidative stress markers in tissue samples if not all cell types are damaged equally. Therefore, a primary goal should be the detection of a stable oxidation product of guanine to insure a reliable detection strategy and for a better understanding of the equilibrium of DNA oxidation and repair.}, subject = {Oxidativer Stress}, language = {de} } @article{RychlikHumpfMarkoetal.2014, author = {Rychlik, Michael and Humpf, Hans-Ulrich and Marko, Doris and D{\"a}nicke, Sven and Mally, Angela and Berthiller, Franz and Klaffke, Horst and Lorenz, Nicole}, title = {Proposal of a comprehensive definition of modified and other forms of mycotoxins including "masked" mycotoxins}, series = {Mycotoxin Research}, volume = {30}, journal = {Mycotoxin Research}, number = {4}, doi = {10.1007/s12550-014-0203-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121240}, pages = {197-205}, year = {2014}, abstract = {As the term "masked mycotoxins" encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, "modified mycotoxins" into "biologically modified" and "chemically modified" with all variations of metabolites of the former and dividing the latter into "thermally formed" and "non-thermally formed" ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term "modified mycotoxins" should be used in the future and the term "masked mycotoxins" to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.}, language = {en} } @article{OttLohseKlotzetal.1982, author = {Ott, Ilka and Lohse, Martin J. and Klotz, Karl-Norbert and Vogt-Moykopf, Ingolf and Schwabe, Ulrich}, title = {Effects of Adenosine on Histamine Release from Human Lung Fragments}, series = {International Archives of Allergy and Immunology}, volume = {98}, journal = {International Archives of Allergy and Immunology}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127877}, pages = {50-56}, year = {1982}, abstract = {The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A 23187 orwith concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl] methyloxy]-phenyl]-1,3-dipropylaxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioninosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence ofXAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a si te antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.}, language = {en} } @article{LohseKlotzSalzeretal.1988, author = {Lohse, Martin J. and Klotz, Karl-Norbert and Salzer, Manfred J. and Schwabe, Ulrich}, title = {Adenosine regulates the \(Ca^{2+} \) sensitivity of mast cell mediator release : (histamine secretion/inositol phosphates/calcium)}, series = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {85}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127883}, pages = {8875-8879}, year = {1988}, abstract = {Mast cells release histamine and other mediators of allergy in response to stimulation of their IgE receptors. This release is generally thought to be mediated by an elevation of cytosolic \(Ca^{2+}\). Recent evidence suggests that there might be factors that modulate the coupling between \(Ca^{2+}\) levels and mediator release. The present report identifies adenosine as one such modulator. Adenosine and several of its metabolically stable analogues were shown to enhance histamine release from rat peritoneal mast cells in response to stimuli such as concanavalin A. Metabolizing endogenous adenosine with adenosine deaminase dampened the response to stimuli, whereas trapping endogenous adenosine inside mast cells with nucleoside-transport inhibitors markedly enhanced stimulated histamine release. The metabolically stable adenosine analogue 5' -(N-ethylcarboxamido)adenosine (NECA) did not affect the initial steps in the sequence from IgE-receptor activation to mediator release, which are generation of inositol trisphosphate and increase of cytosolic \(Ca^{2+}\). However, NECA did enhance the release induced in ATP-permeabilized cells by exogenous \(Ca^{2+}\), but it had no effect on the release induced by phorbol esters. These data suggest that adenosine sensitizes mediator release by a mechanism regulating stimulus-secretion coupling at a step distal to receptor activation and second-messenger generation.}, language = {en} } @article{LohseKlotzUkenaetal.1984, author = {Lohse, M. J. and Klotz, K.-N. and Ukena, D. and Schwabe, U.}, title = {Characterization of \([^3H]\)Phenobarbital Binding to Rat Brain Membranes}, series = {Neuroscience Letters}, volume = {52}, journal = {Neuroscience Letters}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127894}, pages = {97-101}, year = {1984}, abstract = {The binding of \([^3H]\)phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. \((K_d = 700 μM)\) and very high density \((B_{max} = 2.7 nmoll/mg protein)\). It was unaffected by temperature changes from O°C to 95°C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of \([^3H]\) phenobarbital is a rather non-specific interaction with the plasma membrane.}, language = {en} } @article{BoivinBeyersdorfPalmetal.2015, author = {Boivin, Val{\´e}rie and Beyersdorf, Niklas and Palm, Dieter and Nikolaev, Viacheslav O. and Schlipp, Angela and M{\"u}ller, Justus and Schmidt, Doris and Kocoski, Vladimir and Kerkau, Thomas and H{\"u}nig, Thomas and Ertl, Georg and Lohse, Martin J. and Jahns, Roland}, title = {Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by \(Anti-β_1-Adrenoceptor\) Antibodies in a Human-Analogous Rat Model}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {2}, doi = {10.1371/journal.pone.0117589}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126028}, pages = {e0117589}, year = {2015}, abstract = {Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9\% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure.}, language = {en} } @phdthesis{Segerer2019, author = {Segerer, Gabriela}, title = {Characterization of cell biological and physiological functions of the phosphoglycolate phosphatase AUM}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123847}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer. Still, most of the mammalian HAD phosphatases remain functionally uncharacterized. This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos. To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation. The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells. These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling. Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase. To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions. Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20\% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under hypoxic (~1\% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3'-phosphate (GADP). Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism. Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.}, subject = {Phosphoglykolatphosphatase}, language = {en} } @phdthesis{Stumpf2015, author = {Stumpf, Anette D.}, title = {Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125469}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements. For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.}, subject = {Adenosinrezeptor}, language = {en} } @article{CalebiroMaiellaro2014, author = {Calebiro, Davide and Maiellaro, Isabella}, title = {cAMP signaling microdomains and their observation by optical methods}, series = {Frontiers in Cellular Neuroscience}, volume = {8}, journal = {Frontiers in Cellular Neuroscience}, issn = {1662-5102}, doi = {10.3389/fncel.2014.00350}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118252}, pages = {350}, year = {2014}, abstract = {The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.}, language = {en} }