@article{CristalliEleuteriVittorietal.1992, author = {Cristalli, G. and Eleuteri, A. and Vittori, S. and Volpini, R. and Lohse, M. J. and Klotz, Karl-Norbert}, title = {2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamides as selective agonists at A\(_2\) adenosine receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60412}, year = {1992}, abstract = {In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies.}, subject = {Toxikologie}, language = {en} } @article{NolteLorenzenLehretal.1992, author = {Nolte, D. and Lorenzen, A. and Lehr, H.-A. and Zimmer, F.-J. and Klotz, Karl-Norbert and Messmer, K.}, title = {Reduction of postischemic leukocyte-endothelium interaction by adenosine via A\(_2\) receptor}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60424}, year = {1992}, abstract = {The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. leukocytes were stained in vivo with acridine orange (0.5 mg kg\(^{-1}\) min\(^{-1}\) i.v. ). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. ·Adenosine, the adenosine A1-selective agonist 2-chloro-N\(^6\) -cyclopentyladenosine (CCPA), the Arselective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener {XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused viajugular vein starting 15 min priortorelease of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p< 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces postischemic leukocyte-endothelium interaction via A\(_2\) receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.}, subject = {Toxikologie}, language = {en} } @article{UkenaSchirrenKlotzetal.1985, author = {Ukena, D. and Schirren, C. G. and Klotz, Karl-Norbert and Schwabe, U.}, title = {Evidence for an A\(_2\) adenosine receptor in guinea pig lung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60202}, year = {1985}, abstract = {Adenosine receptors in guinea pig lung were characterized by measurement of cyclic AMP formation and radioligand binding. 5'-N-Ethylcarboxamidoadenosine (NECA) increased cyclic AMP Ievels in lung slices about 4-fold over basal values with an EC\(_{50}\) of 0.32 \(\mu\)mol/l. N\(^6\) - R-(- )-Phenylisopropyladenosine (R-PIA) was 5-fold less potent than NECA. 5'-N-Methylcarboxamidoadenosine (MECA) and 2-chloroadenosine had EC\(_{50}\)-values of 0.29 and 2.6 \(\mu\)mol/l, whereas adenosine and inosine had no effect. The adenosine receptors in guinea pig Iung can therefore be classified as A\(_2\) receptors. Several xanthine derivatives antagonized the NECA-induced increase in cyclic AMP levels. 1,3-Diethyl-8-phenylxanthine (DPX; K\(_i\) 0.14 \(\mu\)mol/l) was the most potent analogue, followed by 8-phenyltheophylline (K\(_i\) 0.55 \(\mu\)mol/l), 3-isobutyl-1-methylxanthine (IBMX; K\(_i\) 2.9 \(\mu\)mol/l) and theophylline (K\(_i\) 8.1 \(\mu\)mol/l). In contrast, enprofylline (1 mmol/1) enhanced basal and NECA-stimulated cyclic AMP formation. In addition, we attempted to characterize these receptors in binding studies with [\(^3\)H]NECA. The K\(_D\) for [\(^3\)H] NECA was 0.25 \(\mu\)mol/l and the maximal number of binding sites was 12 pmol/mg protein. In competition experiments MECA (K\(_i\) 0.14 \(\mu\)mol/l) was the most potent inhibitor of [\(^3\)H] NECA binding, followed by NECA (K\(_i\) 0.19 \(\mu\)mol/l) and 2-chloroadenosine (K\(_i\) 1.4 \(\mu\)mol/l). These results correlate well with the EC\(_{50}\)- values for cyclic AMP formation in lung slices. However, the K\(_i\)-values of R-PIA and theophylline were 240 and 270 \(\mu\)mol/l, and DPX and 8-phenyltheophylline did not compete for [\(^3\)H]NECA binding sites. Therefore, a complete characterization of A\(_2\) adenosine receptors by [\(^3\)H] NECA binding was not achieved. In conclusion, our results show the presence of adenylate cyclase-coupled A\(_2\) adenosiile receptors in lung tissue which are antagonized by several xanthines.}, subject = {Toxikologie}, language = {en} } @article{KlotzLohseSchwabeetal.1989, author = {Klotz, Karl-Norbert and Lohse, M. J. and Schwabe, U. and Cristalli, G. and Vittori, S. and Grifantini, M.}, title = {2-Chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) - a high affinity agonist radioligand for A\(_1\) adenosine receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60328}, year = {1989}, abstract = {The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCP A was prepared with a specifi.c radioactivity of 1.58 TBqjmmol ( 43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity K0 -value of 0.2 nmol/1. In the presence of GTP a K0 -value of 13 nmol/1 was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confrrmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgC12, as has been shown for the agonist [ 3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A K0 -value of 0.4 nmol/1 and a Bmax-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmoljl, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [ 3H]CCPA proved to be a useful agonist radioligand for characterization of A 1 adenosine receptors also in tissues with very low receptor density.}, subject = {Toxikologie}, language = {en} } @article{TawfikSchlieperKlotzKreyeetal.1989, author = {Tawfik-Schlieper, H. and Klotz, Karl-Norbert and Kreye, V. A. W. and Schwabe, U.}, title = {Characterization of the K\(^+\)-channel-coupled adenosine receptor in guinea pig atria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60333}, year = {1989}, abstract = {In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on 86Rb + -efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1 ,3-[\(^3\)H]dipropylxanthine ([\(^3\)H]DPCPX) to atrial membrane preparations. The rate of \8^{86}\)Rb\(^+\) -effiux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC50-values for 2-chloro-N\(^6\)-cyclopentyladenosine (CCPA), R-N\(^6\)-phenylisopropyladenosine (R-PIA), 5'-Nethylcarboxamidoadenosine (NECA), and S-N\(^6\)-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 \(\mu\)M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K\(_B\)-value of 8.1 nM, indicating competitive antagonism. [\(^3\)H]DPCPX showed a saturable binding to atrial membranes with a Bmax·value of 227 fmol/mg protein and a K\(_D\)-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K\(^+\) channel-coupled adenosine receptor in guinea pig atria is of an A\(_1\) subtype.}, subject = {Toxikologie}, language = {en} } @article{ShephardLutzSchlatter1994, author = {Shephard, S. E. and Lutz, Werner K. and Schlatter, C.}, title = {The lacI transgenic mouse mutagenicity assay: quantitative evaluation in comparison to tests for carcinogenicity and cytogenetic damage in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60638}, year = {1994}, abstract = {The detection Iimit of the lacl transgenic mouse mutagenicity assay lies, in practice, at approximately a 50-100\% increase in mutant frequency in treated animals over controls. The sensitivity of this assay in detecting genotoxins can be markedly improved by subchronic rather than acute application of the test compound. The lac/ transgenic mouse mutagenicity assay was compared quantitatively to rodent carcinogenicity tests and to presently used in vivo mutagenicity assays. With the genotoxic carcinogens tested thus far, a rough correlation between mutagenic potency and carcinogenic potency was observed: on average, to obtain a doubling in lacl mutant frequency the mice bad to be treated with a total dose equal to 50 times the TD50 daily dose Ievel. This total dose could be administered eilher at a high dose rate within a few days or, preferably, at a low dose rate over several weeks. This analysis also indicated that a lacl experiment using a 250-day exposure period would give a detection Iimit approximately equal to that of a long-term carcinogenicity study. In comparison to the micronucleus test or the chromosome aberration assay, acute sturlies with the presently available lacl system offered no increase in sensitivity. However, subchronic lacl sturlies (3-4-month exposure) resulted in an increase in sensitivity over the established tests by 1-2 orders of magnitude (shown with 2-acetylaminofluorene, N-nitrosomethylamine, N-nitrosomethylurea and urethane). 1t is concluded that a positive result in the lacl test can be highly predictive of carcinogenicity butthat a negative result does not provide a large margin of safety.}, subject = {Toxikologie}, language = {en} } @phdthesis{Hintzsche2011, author = {Hintzsche, Henning}, title = {Gentoxizit{\"a}t nichtionisierender Strahlung - Auswirkungen von Mobilfunk- und Terahertzstrahlung auf das Genom}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-57684}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Ziel der vorliegenden Arbeit war es, zu untersuchen, ob nichtionisierende elektromagnetische Strahlung verschiedener Frequenzbereiche Genomschaden hervorrufen kann. Im Rahmen der vorliegenden Arbeit wurde eine Biomonitoring-Studie zu dieser Thematik konzipiert und durchgef{\"u}hrt. Es wurden 131 Probanden detailliert zu ihrer Mobilfunknutzung befragt. Anschließend wurden Mundschleimhautzellen entnommen und f{\"u}r eine mikroskopische Untersuchung aufbereitet und angef{\"a}rbt. In den Zellen wurden Mikrokerne und andere Kernanomalien quantifiziert. Es zeigte sich keine Erh{\"o}hung der Mikrokernfrequenz in Abh{\"a}ngigkeit von der Dauer der Mobiltelefonnutzung. Auch die anderen abgefragten Parameter hatten keinen Einfluss auf die H{\"o}he des Genomschadens. Als Positivkontrollen wurden vier Patienten, die eine lokale Strahlentherapie (ionisierende Strahlung) erhielten, eingeschlossen. Hier zeigte sich eine deutliche Erh{\"o}hung der Mikrokernfrequenz. Um festzustellen, ob die Mikrokerninduktion erst bei h{\"o}heren Leistungsflussdichten als denen, die beim Mobilfunk verwendet werden, auftritt, wurden in-vitro-Versuche durchgef{\"u}hrt, bei denen verschiedene Zelllinien einer Strahlung von 900 MHz ausgesetzt wurden. Nach Exposition und einer Postinkubationsperiode wurden die Zellen fixiert und die Mikrokernfrequenz bestimmt. Neben den Leistungen wurden hier auch die Expositionszeiten und die Postinkubationsperioden variiert. In keinem Fall konnte eine Erh{\"o}hung der Mikrokernfrequenz festgestellt werden. Insgesamt konnte ein Einfluss elektromagnetischer Strahlung auf das Genom weder am Menschen im Rahmen einer Biomonitoring-Studie noch an verschiedenen Zelllinien im Rahmen von in-vitro-Versuchen festgestellt werden. Terahertzstrahlung ist elektromagnetische Strahlung im Bereich von 0,1 bis 10 THz, d. h. sie liegt zwischen Mikrowellen und Infrarotlicht. Derzeit wird sie haupts{\"a}chlich f{\"u}r spektroskopische Untersuchungen und zur Qualit{\"a}tskontrolle im Herstellungs-prozess verschiedener Produkte verwendet. Anwendungen in der Sicherheitstechnik (z. B. Ganzk{\"o}rperscanner) und in der Medizintechnik (z. B. Bildgebung) stehen kurz vor der Markteinf{\"u}hrung bzw. sind bereits etabliert. Diese Anwendungen bringen eine Exposition der betroffenen Menschen mit sich. Außerdem wird an weiteren Techniken wie etwa der Daten{\"u}bertragung gearbeitet. Die Wirkungen auf biologische Systeme sind im Gegensatz zum Mobilfunkbereich bisher nur unzureichend untersucht. Da bisher keine vollst{\"a}ndigen Literatur{\"u}bersichten vorlagen, wurde eine umfassende Literaturrecherche durchgef{\"u}hrt. Ziel war es, alle bisher durchgef{\"u}hrten Studien zu diesem Thema aufzulisten. Um diese Datenbasis zu verbreitern wurden in-vitro-Versuche bei verschiedenen Frequenzen durchgef{\"u}hrt. Als Strahlungsquellen wurden eine Frequenzvervielfacherkaskade (0,106 THz), ein R{\"u}ckw{\"a}rtswellen-Oszillator (0,380 THz) und ein Ferninfrarot-Laser (2,520 THz) eingesetzt. Die Strahlung wurde in einen modifizierten Inkubator gef{\"u}hrt, so dass die Expositionen bei definierter Temperatur und konstantem CO2-Gehalt durchgef{\"u}hrt werden konnten. Da Terahertzstrahlung durch Wasser sehr stark absorbiert wird, sind bei einer Exposition des Menschen prim{\"a}r die obersten Hautschichten betroffen. Aus diesem Grund wurden prim{\"a}re Hautfibroblasten und HaCaT-Zellen, eine Keratinozyten-Zelllinie, als biologische Systeme verwendet. Die Zellen wurden f{\"u}r unterschiedliche Zeitperioden mit verschiedenen Leistungsflussdichten exponiert. Anschließend wurden die Zellen f{\"u}r den Comet Assay aufbereitet und analysiert. Der Comet Assay ist eine Methode zur Quantifizierung von Einzel- und Doppelstrangbr{\"u}chen der DNA. Weiterhin wurden die Zellen nach einer Postinkubationsperiode f{\"u}r den Mikrokerntest aufbereitet. Neben unbehandelten Kontrollen und Sham-Expositionen wurden auch Positivkontrollen durchgef{\"u}hrt. Es konnte keine Erh{\"o}hung der Anzahl der DNA-Strangbr{\"u}che bzw. der Mikrokernfrequenz festgestellt werden. Da bekannt war, dass im Mobilfunkbereich unter bestimmten Bedingungen St{\"o}rungen der Mitose, nicht aber Erh{\"o}hungen der Mikrokernfrequenz, auftreten, wurden Mitosest{\"o}rungen nach Exposition bei 0,106 THz untersucht. Hierzu wurden AL-Zellen f{\"u}r 30 Minuten exponiert und anschließend ohne Postinkubation direkt fixiert. Analysiert wurden St{\"o}rungen in allen Phasen der Mitose. Es zeigte sich, dass die Frequenz der St{\"o}rungen in der Pro- und Metaphase unver{\"a}ndert blieb. Die St{\"o}rungen in der Ana- und Telophase nahmen dagegen mit steigender Leistungsflussdichte zu. Insgesamt konnte im Terahertzbereich unter den gew{\"a}hlten Expositionsbedingungen kein DNA-Schaden beobachtet werden. Bei 0,106 THz konnten Mitosest{\"o}rungen als Folge der Exposition gezeigt werden. Der Zusammenhang zwischen diesen Mitosest{\"o}rungen und DNA-Sch{\"a}den, insbesondere der Mikrokerninduktion, konnte bisher nicht abschließend gekl{\"a}rt werden und bleibt Gegenstand weiterer Untersuchungen.}, subject = {Mutagenit{\"a}t}, language = {de} } @phdthesis{Bellwon2015, author = {Bellwon, Patricia}, title = {Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122693}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program "Predict-IV"; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of "omics" technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling. This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day. Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors. Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling. Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions. Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo. Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound's distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.}, subject = {Zellkultur}, language = {en} } @article{ChenGassnerBoerneretal.2012, author = {Chen, Wen and Gaßner, Birgit and B{\"o}rner, Sebastian and Nikolaev, Viacheslav O. and Schlegel, Nicolas and Waschke, Jens and Steinbronn, Nadine and Strasser, Ruth and Kuhn, Michaela}, title = {Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway}, series = {Cardiovascular Research}, volume = {93}, journal = {Cardiovascular Research}, number = {1}, doi = {10.1093/cvr/cvr279}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126562}, pages = {141-151}, year = {2012}, abstract = {Aims Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP. Methods and results Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1-/- mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains. Conclusion ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.}, language = {en} } @phdthesis{ZinkgebSondergeld2015, author = {Zink [geb. Sondergeld], Thomas Gerd}, title = {Der Cofilin-Signalweg im Glioblastoma multiforme - Ursachen f{\"u}r den Verlust von Chronophin und Einfluss von LIM-Kinase-Inhibitoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127065}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Das invasive Potential maligner Gliome beeinflusst maßgeblich die schlechte Prognose dieser Tumorentit{\"a}t. Migration und Invasion von Tumorzellen werden entscheidend durch die Cofilin-vermittelte Umstrukturierung des Aktin-Zytoskeletts gepr{\"a}gt, die durch die Aktivit{\"a}t antagonistischer Cofilin-Kinasen und -Phosphatasen reguliert wird. Im Rahmen der vorliegenden Arbeit konnte ein progressiver Expressionsverlust der Cofilin-Phosphatase Chronophin mit ansteigendem Malignit{\"a}tsgrad astrozyt{\"a}rer Gliome aufgezeigt werden, der mit einer Zunahme der Phosphorylierung von Cofilin einhergeht. In den entsprechenden Gewebeproben gelang gleichzeitig der Nachweis einer gesteigerten Expression der Cofilin-Kinase LIMK-2. Genetische und epigenetische Analysen des Chronophin-Locus konnten eine Hypermethylierung im Bereich der Promotorregion der Phosphatase identifizieren, die m{\"o}glicherweise dem Verlust von Chronophin in Glioblastom-Gewebeproben zugrunde liegt. In Glioblastom-Zelllinien, die unterschiedliche Expressionsmuster von Chronophin aufwiesen, konnten hingegen keine molekularen Alterationen festgestellt werden. Untersuchungen des Einflusses von ROCK- und LIMK-Inhibitoren auf Glioblastomzellen konnten ausgepr{\"a}gte Ver{\"a}nderungen der Zellmorphologie dokumentieren, wobei erstmals die Induktion eines stellate cell-Ph{\"a}notyps unter Einfluss des LIMK-Inhibitors BMS-5 beschrieben wird. W{\"a}hrend ROCK- und LIMK-Inhibitoren keinen Einfluss auf die 2D-Motilit{\"a}t der Tumorzellen hatten, wiesen die Glioblastomzellen in Abh{\"a}ngigkeit ihrer basalen Cofilin-Aktivit{\"a}t eine verst{\"a}rkte bzw. verminderte 3D-Invasivit{\"a}t auf. Die Erkenntnisse dieser Arbeit unterstreichen die Bedeutung des Cofilin-Signalweges f{\"u}r die Migration und Invasion von Gliomzellen, zeigen neue Angriffspunkte in der Therapie maligner Gliome auf und warnen zugleich vor einem unkritischen Einsatz neuer Wirkstoffe.}, subject = {Cofilin}, language = {de} }