TY - THES A1 - Neymeyer, Hanna T1 - Annexin A1 im chronischen Nierenversagen T1 - Annexin A1 in chronic renal failure N2 - Die Expansion des renalen Tubulointerstitiums aufgrund einer Akkumulation zellulärer Bestandteile und extrazellulärer Matrix ist eine charakteristische Eigenschaft der chronischen Nierenerkrankung (CKD) und führt zu einer Progression der Erkrankung in Richtung eines terminalen Nierenversagens. Die Fibroblasten Proliferation und ihre Transformation hin zum sekretorischen Myofibroblasten-Phänotyp stellen hierbei Schlüsselereignisse dar. Signalprozesse, die zur Induktion der Myofibroblasten führen, werden aktiv beforscht um anti-fibrotische Therapieansätze zu identifizieren. Das anti-inflammatorische Protein Annexin A1 und sein Rezeptor Formyl-Peptid Rezeptor 2 (FPR2) wurden in verschiedenen Organsystemen mit der Regulation von Fibroblastenaktivität in Verbindung gebracht, jedoch wurden ihre Expression und Funktion bei renalen fibrotischen Erkrankungen bisher nicht untersucht. Ziel der aktuellen Studie war daher die Untersuchung der renalen Annexin A1- und FPR2-Expression in einem Tiermodell des chronischen Nierenversagens, sowie die Charakterisierung der funktionellen Rolle von Annexin A1 in der Regulation des Fibroblasten Phänotyps und ihrer Syntheseleistung. Dazu wurden neugeborene Sprague-Dawley Ratten in den ersten zwei Wochen ihres Lebens entweder mit Vehikel oder mit einem Angiotensin II Typ I Rezeptor Antagonisten behandelt und ohne weitere Intervention bis zu einem Alter von 11 Monaten (CKD Ratten) gehalten. Die Regulation und Lokalisation von Annexin A1 und FPR2 wurden mit Hilfe von Real-Time PCR und Immunhistochemie erfasst. Annexin A1- und FPR2-exprimierende Zellen wurden weiter durch Doppelimmunfluoreszenzfärbungen charakterisiert. Gefärbt wurde mit Antikörpern gegen endotheliale Zellen (rat endothelial cell antigen), Makrophagen (CD 68), Fibroblasten (CD73) und Myofibroblasten (alpha-smooth muscle actin (α-sma)). Zellkulturstudien wurden an immortalisierten renalen kortikalen Fibroblasten aus Wildtyp- und Annexin A1-defizienten Mäusen, sowie an etablierten humanen und murinen renalen Fibrolasten durchgeführt. Eine Überexpression von Annexin A1 wurde durch eine stabile Transfektion erreicht. Die Expression von Annexin A1, α-sma und Kollagen 1α1 wurde durch Real-Time PCR, Western Blot und Immuhistochemie erfasst. Die Sekretion des Annexin A1 Proteins wurde nach TCA-Fällung des Zellkulturüberstandes im Western Blot untersucht. Wie zu erwarten zeigten die CKD Ratten eine geringere Anzahl an Nephronen mit deutlicher glomerulären Hypertrophie. Der tubulointerstitielle Raum war durch fibrilläres Kollagen, aktivierte Fibroblasten und inflammatorische Zellen expandiert. Parallel dazu war die mRNA Expression von Annexin A1 und Transforming growth factor beta (TGF-β) signifikant erhöht. Die Annexin A1-Lokalisation mittels Doppelimmunfluorsezenz identifizierte eine große Anzahl von CD73-positiven kortikalen Fibroblasten und eine Subpopulation von Makrophagen als Annexin A1-positiv. Die Annexin A1-Menge in Myofibroblasten und renalen Endothelien war gering. FPR2 konnte in der Mehrzahl der renalen Fibroblasten, in Myofibroblasten, in einer Subpopulation von Makrophagen und in renalen Epithelzellen nachgewiesen werden. Eine Behandlung der murinen Fibroblasten mit dem pro-fibrotischen Zytokin TGF-β führte zu einem parallelen Anstieg der α-sma-, Kollagen 1α1- und Annexin A1-Biosynthese und zu einer gesteigerten Sekretion von Annexin A1. Eine Überexpression von Annexin A1 in murinen Fibroblasten reduzierte das Ausmaß der TGF-β induzierten α-sma- und Kollagen 1α1-Biosynthese. Fibroblasten aus Annexin A1-defizienten Mäusen zeigten einen starken Myofibroblasten-Phänotyp mit einer gesteigerten Expression an α-sma und Kollagen 1α1. Der Einsatz eines Peptidantagonisten des FPR2 (WRW4) resultierte in einer Stimulation der α-sma-Biosynthese, was die Vermutung nahe legte, dass Annexin A1 FPR2-vermittelt anti-fibrotische Effekte hat. Zusammenfassend zeigen diese Ergebnisse, dass renale kortikale Fibroblasten eine Hauptquelle des Annexin A1 im renalen Interstitium und einen Ansatzpunkt für Annexin A1-Signalwege in der Niere darstellen. Das Annexin A1/FPR2-System könnte daher eine wichtige Rolle in der Kontrolle des Fibroblasten Phänotyp und der Fibroblasten Aktivität spielen und daher einen neuen Ansatz für die anti-fibrotischen pharmakologischen Strategien in der Behandlung des CKD darstellen. N2 - Expansion of the renal tubulointerstitium due to an accumulation of cellular constituents and extracellular matrix is a characteristic feature of chronic kidney disease (CKD) and leads to the progression towards renal failure. Fibroblast proliferation and transformation to the secretory myofibroblast phenotype present key events herein. The signaling process which leads to the generation of myofibroblasts is actively investigated to identify targets for antifibrotic therapeutic strategies. The antiinflammatory protein annexin A1 and its receptor formyl peptide receptor 2 (FPR2) have been implicated in the regulation of fibroblasts from various organs but the expression and function of the two products in renal fibrotic disease have not been elucidated so far. Aim of the present study was therefore to investigate the renal expression of annexin A1 and FPR2 in an animal model of chronic kidney disease and to characterize the role of annexin A1 in the regulation of fibroblast phenotype and synthetic activity. To this end, newborn Sprague-Dawley rats were treated either with vehicle or with an angiotensin II type I receptor antagonist during the first two weeks of their life and kept without further intervention until the age of 11 month (CKD rats). Regulation and localization of annexin A1 and FPR2 were studied using real-time PCR and immunohistochemistry. Annexin A1 and FPR2 expressing cells were further characterized by double labeling immunofluorescence with markers for endothelial cells (rat endothelial cell antigen), macrophages (CD68), fibroblasts (CD73), and myofibroblasts (alpha-smooth muscle actin (α-sma)). Cell culture studies were conducted in immortalized renal cortical fibroblast derived from wildtype and from annexin A1-deficient mice as well as in established cell lines of human and murine renal fibroblasts. Overexpression of annexin A1 was achieved by stable transfection. Expression of annexin A1, α-sma and collagen 1α1 was determined using real-time PCR, Western blotting and immunohistochemistry. Secretion of annexin A1 was studied using trichloroacetic acid protein precipitation of cell culture supernatants and Western blotting. As expected, CKD rats had an overall lower number of nephrons with a marked glomerular hypertrophy. The tubulointerstitial space was expanded due to an accumulation of fibrillar collagens, activated fibroblasts and inflammatory cells. In parallel, mRNA expression for Annexin A1 and transforming growth factor beta (TGF-β) was significantly increased. Double labeling immunofluorescence localization of annexin A1 demonstrated a high abundance in CD73 positive cortical interstitial fibroblasts and in a subset of CD68 immunoreactive macrophages. The abundance in myofibroblasts and renal endothelia was low. FPR2 was found in the majority of renal fibroblasts, myofibroblasts, a subset of macrophages, and in renal endothelial cells. Treatment of cultured murine fibroblasts with the profibrotic cytokine TGF-β resulted in a parallel induction of α-sma-, collagen 1α1- and annexin A1 biosynthesis. In addition, annexin A1 secretion was markedly increased. Overexpression of annexin A1 in murine fibroblasts reduced TGF β-induced α-sma- and collagen 1α1-biosynthesis. Fibroblasts derived from annexin A1-deficient mice showed a strong myofibroblast phenotype with increased expression of both, α-sma-, and collagen 1α1. Application of a peptide antagonist of FPR2 receptor (WRW4) caused a stimulation of α-sma biosynthesis thus suggesting a role of FPR2 in the antifibrotic effects of annexin A1. In conclusion, these results identify renal cortical interstitial fibroblasts as major source and as a target for annexin A1 signalling in the kidney. The annexin A1/FPR2 signalling system may therefore play an important role in the control of fibroblast phenotype and activity and may therefore provide a novel target for antifibrotic pharmacological strategies in the treatment of CKD. KW - Myofibroblasten KW - Transforming Growth Factor beta KW - Formyl-Peptid Rezeptor 2 KW - extrazelluläre Matrix KW - myofibroblast KW - transforming growth factor beta KW - formyl peptide receptor 2 KW - extracellular matrix Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-69670 ER - TY - GEN A1 - Mazurek, Nicole T1 - Untersuchungen zur Genexpression und Differenzierung muriner embryonaler Stammzellen in vitro zur Prädiktion eines embryotoxischen Potentials ausgewählter Chemikalien T1 - Investigations for gene expression and differentiation of murine embryonic stem cells in vitro to predict the embryotoxic potential of selected chemicals N2 - Der Embryonale Stammzelltest (EST) ist ein validierter In-vitro-Embryotoxizitätstest, der zur Untersuchung embryotoxischer Wirkungen von Chemikalien eingesetzt werden kann. Während des zehntägigen Differenzierungsassays differenzieren sich die pluripotenten murinen embryonalen Stammzellen (ES-Zellen) der Linie D3 in vitro in spontan kontrahierende Herzmuskelzellen. Dabei rekapitulieren sie Prozesse der frühen Embryogenese in vivo. Ein Zytotoxizitätsassay mit D3-Zellen und ausdifferenzierten, adulten 3T3-Maus-Fibroblasten dient der Ermittlung allgemeiner zytotoxischer Effekte und unterschiedlicher Sensitivitäten beider Zelllinien. Somit basiert der EST auf den beiden wichtigsten Mechanismen pränataler Toxizität, der Störung der Differenzierung und der Zytotoxizität. Ziel dieser Arbeit war es, mit Hilfe des EST das embryotoxische Potential der vier Chemikalien Trichostatin A (TSA), Methylazoxymethanolacetat (MAMac), Natriumdodecylsulfat (SDS) und Benzoesäure (BA) abzuschätzen. Dazu wurde mikroskopisch ermittelt, bei welcher Testsubstanzkonzentration in 50 % der während der In-vitro-Differenzierung gebildeten Embryonalkörperchen die Kardiomyozytendifferenzierung inhibiert wird (ID50). Außerdem wurde die halbmaximale Hemmkonzentration des Zellwachstums auf die beiden Zelllinien bestimmt (IC50D3 bzw. IC503T3). Als Erweiterung dieses konventionellen EST wurden mittels quantitativer Real Time-PCR an den Tagen 5, 7 und 10 der Differenzierung zusätzlich Genexpressionsanalysen etablierter herzmuskelspezifischer Markergene (Mesoderm Posterior 1, Tag 5; Myosin light chain 1, Tag 7 und 10) durchgeführt. Deren Expression korreliert in den ES-Zellen mit der embryonalen Herzdifferenzierung in vivo und kann zur Ermittlung der von der Prüfsubstanz hervorgerufenen halbmaximalen Hemmung der Genexpression in den Kardiomyozyten (IC50 Exp) herangezogen werden. Um letztlich embryotoxische Effekte in vivo auf Grundlage der ermittelten In-vitro-Daten abschätzen zu können, wurden die ermittelten Parameter mittels eines für den EST empirisch abgeleiteten mathematischen Prädiktionsmodells (PM) zur Klassifizierung der Testsubstanzen als nicht, schwach oder stark embryotoxisch herangezogen. Für jede der Substanzen waren die ermittelten Halbhemmkonzentrationen in den überwiegenden Fällen vergleichbar und führten unter Verwendung des PMs im konventionellen und im molekularen EST zu deren identischer Klassifizierung. TSA wurde als „stark embryotoxisch“ klassifiziert und beeinflusste insbesondere das Differenzierungspotential der ES-Zellen. Das als „schwach embryotoxisch“ klassifizierte SDS wirkte auf die D3-Zellen stärker differenzierungsinhibierend als zytotoxisch, hemmte jedoch das Wachstum der 3T3-Zellen bereits in deutlich niedrigeren Konzentrationen. MAMac und BA wurden als „nicht embryotoxisch“ klassifiziert. Bei ihnen stand die zytotoxische Wirkung deutlich im Vordergrund. Diese Prädiktionen stimmten mit In-vivo-Befunden überein, was von der Stabilität und der Brauchbarkeit der im konventionellen und molekularen EST ermittelten Parameter zeugte. Einzige Ausnahme war das als Entwicklungsneurotoxin in vivo bekannte MAMac. Da der EST auf mesodermaler Differenzierung basiert, können spezifische Effekte auf neuronale Entwicklungsprozesse offenbar nicht vollständig erfasst werden. Substanzkonzentrationen, die sich als differenzierungsinhibierend auf die morphologische Kardiomyozytendifferenzierung erwiesen haben, führten auch zu einer messbaren Repression der herzmuskelspezifischen Genexpression. Dabei erwies sich die IC50 Exp als ebenso sensitiv wie die konventionellen Parameter und als nutzbringende Ergänzung zu diesen, da sie bereits nach 5 bzw. 7 Tagen der In-vitro-Differenzierung eine mit dem mikroskopischen Parameter übereinstimmende Einschätzung des embryotoxischen Potentials der Chemikalien in vivo ermöglichte. Genexpressionsanalysen weiterer differenzierungsspezifischer Gene können zusätzlich zur Aufklärung zu Grunde liegender Mechanismen der Embryotoxizität von Testsubstanzen dienen. Somit kann der EST durch die Vorteile der Stammzelltechnologie und der Genexpressionsanalyse als neues prädiktives Screening-Instrument zur frühzeitigen Detektion embryotoxischer Substanzeffekte in der pharmazeutischen und chemischen Industrie genutzt werden. N2 - The embryonic stem cell test (EST) represents a validated in vitro embryotoxicity test that can be utilised for investigations of embryotoxic effects of chemical substances. During the 10-day differentiation assay the pluripotent murine embryonic stem cells (ES cells) of the D3 line differentiate in vitro into spontaneously beating cardiac muscle cells that can be observed microscopically. Thereby, ES cells recapitulate processes of early embryogenesis in vivo. A cytotoxicity assay with D3 cells as well as differentiated, adult 3T3 mouse fibroblasts is used to determine general cytotoxic effects and to consider differences in the sensitivity of both cell lines. Hence the EST is based on the two most important mechanisms of prenatal toxicity, such as inhibition of differentiation and cytotoxicity. The aim of the presented work consisted in the evaluation of the embryotoxic potential of the four chemicals trichostatin A (TSA), methylazoxymethanolacetate (MAMac), sodium dodecyl sulfate (SDS) and benzoic acid (BA) by means of the EST. For this purpose the concentration of the test substance that causes an inhibition of cardiomyocyte differentiation in 50 % of the embryoid bodies which are formed during the in vitro differentiation (ID50-value) and the halfmaximal inhibiting concentration of cell proliferation of D3 and 3T3 cell lines (IC50D3 and IC503T3) were determined. As extension of this conventional EST, the effect of test substances was investigated at the molecular level by gene expression analyses of cardiac specific genes (Mesoderm Posterior 1, day 5; Myosin light chain 1, day 7 and 10). Their expression in ES cells correlates with the embryonic heart differentiation in vivo. Quantitative Real Time-PCR gene expression analysis was used to determine the halfmaximal inhibition of the cardiomyocyte gene expression (IC50 Exp) caused by the test compound. To predict embryotoxic effects in vivo from the determined in vitro data, these parameters were used for the classification of the test chemicals as non, weak or strong embryotoxic via a mathematical prediction model (PM). In the majority of cases comparable halfmaximal inhibiting concentrations were calculated in the conventional and molecular EST that resulted in the identical classification of the tested chemicals concerning their embryotoxic potential. TSA was estimated as “strongly embryotoxic” and affected particularly the differentiation potential of the ES cells. SDS was classified as “weakly embryotoxic” and acted by inhibiting the differentiation of D3 cells at concentrations lower than cytotoxic concentrations but already repressed the growth of the 3T3 cells in significantly lower ranges. As to MAMac and BA that were classified as “non-embryotoxic” the cytotoxic effects on both cell lines predominated. These predictions were consistent with in vivo findings that testifies the stability and the usefulness of the parameters used in the conventional and molecular EST. MAMac, which is known as a developmental neurotoxin in vivo, represented the single exception. Its misclassification as compared to in vivo data may originate from the limitations of the model system that is based on mesodermal differentiation. Thus, specific effects on neuronal developmental processes obviously cannot be detected completely. Gene expression analysis showed that test substance concentrations which were proved to be inhibiting on the morphological differentiation of cardiomyocytes caused a repression of cardiac-specific marker gene expression as well. Thereby, IC50 Exp-values proved to be just as sensitive as the conventional parameters and can provide valuable and supportive data. They allowed a prediction of the embryotoxic potential of the chemicals in vivo already at day 5 and day 7 of in vitro differentiation. Moreover, gene expression analysis of appropriate differentiation specific genes could be used to investigate mechanisms that are responsible for embryotoxic properties of the test compounds. Thus, the EST is considered to represent a new, predictive screening test especially in the pharmaceutical industry to detect the embryotoxic potential of chemical compounds early in the process of compound development. KW - Embryonaler Stammzelltest (EST) KW - D3-Zellen KW - Differenzierungsassay KW - Zytotoxizitätsassay KW - Genexpressionsanalysen (qRT-PCR) KW - embryonic stem cell test (EST) KW - D3 cells KW - differentiation assay KW - cytotoxicity assay KW - gene expression analysis (qRT-PCR) Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-68912 ER - TY - THES A1 - Schumann, Sara T1 - Influence of intestinal inflammation on bacterial protein expression in monoassociated mice T1 - Der Einfluss chronisch-entzündlicher Darmerkrankungen auf die Proteinexpression intestinaler E. coli N2 - Background: Increased numbers of intestinal E. coli are observed in inflammatory bowel disease, but the reasons for this proliferation and it exact role in intestinal inflammation are unknown. Aim of this PhD-project was to identify E. coli proteins involved in E. coli’s adaptation to the inflammatory conditions in the gut and to investigate whether these factors affect the host. Furthermore, the molecular basis for strain-specific differences between probiotic and harmful E. coli in their response to intestinal inflammation was investigated. Methods: Using mice monoassociated either with the adherent-invasive E. coli (AIEC) strain UNC or the probiotic E. coli Nissle, two different mouse models of intestinal inflammation were analysed: On the one hand, severe inflammation was induced by treating mice with 3.5% dextran sodium sulphate (DSS). On the other hand, a very mild intestinal inflammation was generated by associating interleukin 10-deficient (IL-10-/-) mice with E. coli. Differentially expressed proteins in the E. coli strains collected from caecal contents of these mice were identified by two-dimensional fluorescence difference gel electrophoresis. Results DSS-experiment: All DSS-treated mice revealed signs of a moderate caecal and a severe colonic inflammation. However, mice monoassociated with E. coli Nissle were less affected. In both E. coli strains, acute inflammation led to a downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower caecal concentrations of bacterial fermentation products than the control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterised protein YggE. NfuA was upregulated nearly 3-fold in both E. coli strains after DSS administration. Reactive oxygen species produced during intestinal inflammation damage Fe-S clusters and thereby lead to an inactivation of Fe-S proteins. In vitro data indicated that the repair of Fe-S proteins by NfuA is a central mechanism in E. coli to survive oxidative stress. Expression of YggE, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC under control and inflammatory conditions. In vitro growth experiments confirmed these results, indicating that E. coli Nissle is better equipped to cope with oxidative stress than E. coli UNC. Additionally, E. coli Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold higher than E. coli UNC. In turn, caecal indole concentrations resulting from cleavage of tryptophan by TnaA were higher in E. coli Nissle- associated control mice than in the respective mice associated with E. coli UNC. Because of its anti-inflammatory effect, indole is hypothesised to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle. Results IL-10-/--experiment: Only IL-10-/- mice monoassociated with E. coli UNC for 8 weeks exhibited signs of a very mild caecal inflammation. In agreement with this weak inflammation, the variations in the bacterial proteome were small. Similar to the DSS-experiment, proteins downregulated by inflammation belong mainly to the central energy metabolism. In contrast to the DSS-experiment, no upregulation of chaperone proteins and NfuA were observed, indicating that these are strategies to overcome adverse effects of strong intestinal inflammation. The inhibitor of vertebrate C-type lysozyme, Ivy, was 2- to 3-fold upregulated on mRNA and protein level in E. coli Nissle in comparison to E. coli UNC isolated from IL-10-/- mice. By overexpressing ivy, it was demonstrated in vitro that Ivy contributes to a higher lysozyme resistance observed for E. coli Nissle, supporting the role of Ivy as a potential fitness factor in this E. coli strain. Conclusions: The results of this PhD-study demonstrate that intestinal bacteria sense even minimal changes in the health status of the host. While some bacterial adaptations to the inflammatory conditions are equal in response to strong and mild intestinal inflammation, other reactions are unique to a specific disease state. In addition, probiotic and colitogenic E. coli differ in their response to the intestinal inflammation and thereby may influence the host in different ways. N2 - Hintergrund: Chronisch entzündliche Darmerkrankungen zeichnen sich unter anderem durch eine starke Proliferation intestinaler E. coli aus. Unbekannt ist jedoch, ob diese Vermehrung eine Ursache oder eine Folge der Erkrankung darstellt. Ziel der vorliegenden Doktorarbeit war es daher, E. coli-Proteine zu identifizieren, welche der Anpassung an die entzündlichen Bedingungen im Darmtrakt dienen und unter Umständen einen Effekt auf den Gesundheitszustand des Wirtes haben. Weiterhin sollten die molekularen Ursachen für stammesspezifische Unterschiede zwischen probiotischen und gesundheitsschädlichen E. coli näher untersucht werden. Methoden: In den tierexperimentellen Analysen wurden keimfreie Mäuse entweder mit dem probiotischen E. coli Nissle oder dem adhärent-invasiven E. coli UNC monoassoziiert und in zwei verschiedenen Entzündungsmodellen näher untersucht. Einerseits wurde eine starke Darmentzündung durch die Gabe von 3,5% Natrium-Dextransulfat (DSS) ausgelöst. Andererseits wurde in Interleukin 10-defizienten (IL-10-/-) Mäusen eine sehr milde Form der Entzündung durch Besiedlung mit E. coli induziert. Die E. coli Bakterien wurden am Ende der Versuche aus den Caecuminhalten der Mäuse isoliert und die bakterielle Proteinexpression wurde mittels zwei-dimensionaler Gelelektrophorese analysiert. Ergebnisse des DSS-Versuchs: Alle Tiere des DSS-Versuchs entwickelten unabhängig vom E. coli Stamm, mit dem sie besiedelt waren, eine moderate Entzündung im Caecum und eine starke im Colon, wobei die Entzündungsreaktion durch die Monoassoziation mit E. coli Nissle leicht abgeschwächt wurde. In beiden E. coli Stämmen führte die Darmentzündung zu einer verringerten Expression von Enzymen des Kohlenhydratabbaus und der Energiegewinnung. In Folge dessen waren die intestinalen Konzentrationen bakterieller Fermentationsprodukte in den entzündeten Tieren geringer als in den gesunden Kontrolltieren. Weitere differentiell exprimierte Proteine umfassen das Fe-S- Cluster Reparaturprotein NfuA, die Tryptophanase TnaA und das uncharakterisierte Protein YggE. In beiden E. coli Stämmen, welche aus den DSS-Tieren isoliert wurden, war das NfuA Protein dreifach höher exprimiert. Eine Darmentzündung führt zu einer vermehrten Bildung reaktiver Sauerstoffspezies, welche die Fe-S-Cluster in Eisen-Schwefel-Proteinen zerstören und damit zu einer Inaktivierung dieser Proteine führen. In vitro Untersuchungen bestätigten, dass die Reparatur der Eisen-Schwefel-Proteine durch NfuA ein wichtiger Mechanismus ist um oxidativem Stress entgegenzuwirken. Das YggE Protein, welches laut Literaturangaben einen hemmenden Einfluss auf die Bildung reaktiver Sauerstoffspezies hat, war in E. coli Nissle 4- bis 8-fach erhöht (verglichen mit E. coli UNC unter Kontroll- und Entzündungsbedingungen). In vitro Versuche bestätigten diese Daten und zeigten, dass E. coli Nissle im Vergleich zu E. coli UNC eine erhöhte Resistenz gegenüber oxidativem Stress aufweist. Außerdem wurde im Vergleich E. coli Nissle vs. E. coli UNC (unter Entzündungs- und Kontrollbedingungen) ein 4- bis 7-fach erhöhter TnaA-Gehalt nachgewiesen. Indol, das Produkt der TnaA-katalysierten Tryptophanspaltung wurde in erhöhten Mengen im Intestinaltrakt E. coli Nissle-assoziierter Kontrolltiere detektiert. Seit längerem werden entzündungshemmende Eigenschaften für Indol postuliert, die aufgrund der Ergebnisse dieser Doktorarbeit nun auch mit den gesundheitsfördenden Eigenschaften von E. coli Nissle in Zusammenhang gebracht werden können. Ergebnisse des IL-10-/-- Versuchs: Nach einer 8-wöchigen Assoziationsdauer wurde nur in den mit E. coli UNC besiedelten IL-10-/- Tieren eine schwache Entzündungsreaktion nachgewiesen. Bedingt durch diese sehr schwach ausgeprägte Entzündungsantwort waren auch die Veränderungen im bakteriellen Proteom von E. coli UNC nur gering. Wie im DSS-Versuch waren Proteine des bakteriellen Energiestoffwechsels reprimiert, allerdings wurde keine Induktion von NfuA beobachtet. Daher scheint die Induktion von NfuA nur der Anpassung an eine starke Entzündung zu dienen. Weiterhin wurde nachgewiesen, dass E. coli Nissle aus IL-10-/- Tieren den Hemmer für das vertebrate C-Typ Lysozym (Ivy) sowohl auf mRNA- als auch auf Proteinebene stärker exprimiert als E. coli UNC. Überexpression von Ivy unter in vitro Bedingungen zeigte, dass es an der erhöhten Lysozymresistenz von E. coli Nissle beteiligt ist und somit eine Rolle als möglicher Fitnessfaktor von E. coli Nissle spielt. Schlussfolgerungen: In dieser Doktorarbeit wurde gezeigt, dass Darmentzündungen die Proteinexpression eines im Darm lebenden Bakteriums beeinflussen. Einige der aufgedeckten bakteriellen Anpassungsreaktionen werden sowohl bei einer starken als auch bei einer schwachen Entzündung ausgelöst; andere wiederum sind spezifisch für nur einen dieser Entzündungszustände. Weiterhin wurde deutlich, dass sich E. coli-Stämme hinsichtlich ihrer Reaktion auf eine Darmentzündung unterscheiden und damit möglicherweise den Wirt beeinflussen.  KW - E. coli KW - chronisch-entzündliche Darmerkrankungen KW - Proteom KW - Ivy KW - Probiotika KW - E. coli KW - inflammatory bowel disease KW - proteomics KW - Ivy KW - probiotics Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-67757 ER - TY - GEN A1 - Gardemann, Andreas A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt T1 - Nervous control of liver metabolism and hemodynamics N2 - Content: Anatomy of hepatic innervation In vivo studies on the role of hepatic nerves Effects of hepatic nerves in isolated perfused liver Mechanism of action of sympathetic hepatic nerves T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 164 Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-51346 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt T1 - Integration of function in the hepatic acinus : intercellular communication in neural and humoral control of liver metabolism N2 - Content: Architecture of the liver acinus Functional zonation of the liver acinus Topological organization of metabollc regulation in the acinus Topological organization of defense and organ structure regulation in the acinus T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 163 Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-51279 ER - TY - GEN A1 - Hespeling, Ursula A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt A1 - Götze, Otto A1 - Zwirner, Jörg T1 - Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co-cultures N2 - Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 pg/ml increased the formation of thromboxane A₂, prostaglandin D₂, E₂ and F₂α6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 μM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 117 Y1 - 1995 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45909 ER - TY - GEN A1 - Muschol, Waldemar A1 - Püschel, Gerhard Paul A1 - Hülsmann, Martina A1 - Jungermann, Kurt T1 - Eicosanoid-mediated increase in glucose and lactate output as well as decrease and redistribution of flow by complement-activated rat serum in perfused rat liver N2 - Rat serum, in which the complement sytem had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4-chlorobenzenesulfonamide)-ethyl]-benzene-acetica cid (BM 13505), but clearly less efficiently by the 5’-lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-{3-[3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-propoxy]-4-chlorine-6-methyl-phenyl}-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B₂ and prostaglandin F₂α into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - pape 116 Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45892 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Miura, Hisayuki A1 - Neuschäfer-Rube, Frank A1 - Jungermann, Kurt T1 - Inhibition by the protein kinase C activator 4β-phorbol 12-myristate 13-acetate of the prostaglandin F₂α-mediated and noradrenaline-mediated but not glucagon-mediated activation of glycogenolysis in rat liver N2 - In perfused rat livers, infusion of prostaglandin F₂α (PGF₂α) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13-myristate 14-acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF₂α but did not alter the effects of glucagon. In isolated rat hepatocytes PGF₂α, noradrenaline and glucagon activated glycogen phosphorylase but only PGF₂α and noradrenaline increased intracellular inositol 1,4,5-1risphosphalc (InsP₃). The noradrenaline- or PGF₂α-elicited activation of glycogen phosphorylase and increase in InsP₃ were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon-mediated enzyme activation was not affected. In contra\t to PMA, the phorbol ester 4a-phorbol 13,14-didecanoate. which does not activate protein kinase C, did not attenuate the PGF₂α- and noradrenaline-elicited stimulation of glucose output, glycogen phosphorylase and InsP, formation. Stimulation of InsP₃ formation by AlF₄⁻, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA. Plasma membranes purified from isolated hepatocytes had both a high-capacity, low-affinity and a low-capacity, high-affinity binding site for PGF₂α. The Kd of the high-capacity, low-affinity binding site was close to the concentration of PGF₂α that increased glycogen phosphorylase activity halfmaximally. Binding to the high-capacity, low-affinity binding site was enhanced by guanosine 5'- 0-(3-thio)triphosphate (GTP[S]). This high-capacity, low-affinity site might thus represent the receptor. The Bmax and Kd of the high-capacity site, as well as the enhancement by GTP[S] of PGF₂α binding to this site, remained unaffected by PMA pretreatment. It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP₃-mediated signal pathway from PGF₂α via a PGF₂α receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 115 Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45889 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Mentlein, Rolf A1 - Heymann, Eberhard T1 - Isolation and characterization of Dipeptidyl Peptidase IV from human placenta N2 - Human placenta is surprisingly rich in post-proline dipeptidyl peptidase activity. Among various cell fractions, microsomes have the highest specific activity. A homogeneous enzyme preparation is obtained in a six-step purification procedure. The final preparation appears homogeneous upon dodecyl sulfate electrophoresis, but analytical isoelectric focussing reveals various active bands with isoelectric points in the range of pH 3 - 4. The enzyme is a glycoprotein containing about 30% carbohydrate. Treatment with neuraminidase lowers the isoelectric points but does not reduce the heterogeneity of the band pattern. The subunit molecular weight is 120000 as estimated by dodecyl sulfate electrophoresis, whereas Mr of the native enzyme is > 200000, as can be concluded from gel filtration experiments. The purified dipeptidyl peptidase cleaves various synthetic and natural peptides, including substance P, kentsin, casomorphin and a synthetic renin inhibitor. In general, the specificity of the placenta peptidase is similar to that of post-proline dipeptidyl peptidase from other sources. Phenylalanylprolyl-P-naphthylamide (Km = 0.02 mM, I/ = 92 Ujmg) is the best substrate among various synthetic peptide derivatives. Only peptides with a free N-terminal amino group and proline, hydroxyproline, or alanine in position 2 of the N-terminal sequence are cieaved. However, X-Pro-Pro- . . . structures, e. g. as in bradykinin, are not attacked. 1 mM bis-(6nitrophenyI)phosphate or 1 mM diisopropylfluorophosphate completely inactivate the peptidase within 30 min at 30°C (pH 8). The peptidase is also completely inhibited by 1 mM Zn²⁺ and by other heavy metals. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 114 Y1 - 1982 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45875 ER - TY - GEN A1 - Neuschäfer-Rube, Frank A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt T1 - Characterization of prostaglandin-F₂α-binding sites on rat hepatocyte plasma membranes N2 - Prostaglandin (PG)F₂α has previously been shown to increase glucose output from perfused livers and isolated hepatocytes, where it stimulated glycogen phosphorylase via an inositol-trisphosphatedependent signal pathway. In this study, PGF₂α binding sites on hepatocyte plasma membranes, that might represent the putative receptor, were characterized. Binding studies could not be performed with intact hepatocytes, because PGF₂α accumulated within the cells even at 4°C. The intracellular accumulation was an order of magnitude higher than binding to plasma membranes. Purified hepatocyte plasma membranes had a high-affinity/low-capacity and a low-affinity/highcapacity binding'site for PGF₂α. The respective binding constants for the high-affinity site were Kd = 3 nM and Bmax = 6 fmol/mg membrane protein, and for the low-affinity site Kd = 426 nM and Bmax = 245 fmol/mg membrane protein. Specific PGF₂α binding to the low-affinity site, but not to the high-affinity site, could be enhanced most potently by GTP[γS] followed by GDP[ϐS] and GTP, but not by ATP[γS] or GMP. PGF₂α competed most potently with [³H]PGF₂α for specific binding to hepatocyte plasma membranes, followed by PGD₂ and PGE₂. Since the low-affinity PGF₂α-binding site had a Kd in the concentration range in which PG had previously been shown to be half-maximally active, and since this binding site showed a sensitivity to GTP, it is concluded that it might represent the receptor involved in the PGF₂α signal chain in hepatocytes. A biological function of the high-affinity site is currently not known. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 113 Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45863 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Kirchner, C. A1 - Schröder, A. A1 - Jungermann, Kurt T1 - Glycogenolytic and antiglycogenolytic prostaglandin E₂ actions in rat hepatocytes are mediated via different signalling pathways N2 - Prostaglandin E₂ has been reported both to stimulate glycogen-phosphorylase activity (glycogenolytic effect) and to inhibit the glucagon-stimulated glycogen-phosphorylase activity (antiglycogenolytic effect) in rat hepatocytes. It was the purpose of this study to resolve this apparent contradiction and to characterize the signalling pathways and receptor subtypes involved in the opposing prostaglandin E₂ actions. Prostaglandin E₂ (10 μM) increased glucose output, glycogen-phosphorylase activity and inositol trisphosphate formation in hepatocyte cell culture andor suspension. In the same systems, prostaglandin E₂ decreased the glucagon-stimulated (1 nM) glycogen-phosphorylase activity and cAMP formation. The signalling pathway leading to the glycogenolytic effect of PGE₂ was interrupted by incubation of the hepatocytes with 4P-phorbol 12-myristate 13-acetate (100 nM) for 10 min, while the antiglycogenolytic effect of prostaglandin E₂ was not attenuated. The signalling pathway leading to the antiglycogenolytic effect of prostaglandin E₂ was interrupted by an incubation of cultured hepatocytes with pertussis toxin (100 ng/ml) for 18 h, whereas the glycogenolytic effect of prostaglandin E₂ was enhanced. The EP₁/EP₃ prostaglandin-E₂-receptor-specific prostaglandin E₂ analogue Sulproston had a stronger glycogenolytic potency than the EP₃ prostaglandin-E₂-receptor-specific prostaglandin E₂ analogue Misoprostol. The antiglycogenolytic potency of both agonists was equal. It is concluded that the glycogenolytic and the antiglycogenolytic effects of prostaglandin E₂ are mediated via different signalling pathways in hepatocytes possibly involving EP₁ and EP₃ prostaglandin E₂ receptors, respectively. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 112 Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45853 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt T1 - Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver N2 - In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by α-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 μM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 110 Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45846 ER - TY - GEN A1 - Neuschäfer-Rube, Frank A1 - DeVries, Christa A1 - Hänecke, Kristina A1 - Jungermann, Kurt A1 - Püschel, Gerhard Paul T1 - Molecular cloning and expression of a prostaglandin E₂ receptor of the EP₃ϐ subtype from rat hepatocytes N2 - Rat hepatocytes have previously been reported to possess prostaglandin E₂ receptors of the EP₃-type (EP₃-receptors) that inhibit glucagonstimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP₃ϐ receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95% homology to the EP₃ϐ receptor from mouse mastocytoma. The amino- and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE₂ with an apparent Kd of 15 nM. PGE₂ > PGF₂α > PGD₂ competed for [³H]PGE₂ binding sites as did the EP₃ receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP₁ receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE₂ > misoprostol > PGF₂α inhibited the forskolin-elicited cAMP formation. Thus, the characteristics of the EP₃ϐ receptor of rat hepatocytes closely resemble those of the EP₃ϐ receptor of mouse mastocytoma. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 108 KW - Prostaglandin receptor KW - Hepatocyte (rat) KW - Molecular cloning and expression Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45830 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Christ, Bruno T1 - Inhibition by PGE₂ of glucagon-induced increase in phosphoenolpyruvate carboxykinase mRNA and acceleration of mRNA degradation in cultured rat hepatocytes N2 - In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E₂ has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE₂ to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE₂ of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE₂ accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE₂ is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE₂ may reduce the hepatic gluconeogenic capacity via a Gᵢ-linked signal chain. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 045 KW - Prostaglandin E₂ KW - Glucagon KW - Phosphoenolpyruvate carboxykinase KW - Inflammation KW - mRNA degradation Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45792 ER - TY - GEN A1 - Watanabe, Yuji A1 - Püschel, Gerhard Paul A1 - Gardemann, Andreas A1 - Jungermann, Kurt T1 - Presinusoidal and proximal intrasinusoidal confluence of hepatic artery and portal vein in rat liver : functional evidence by orthograde and retrograde bivascular perfusion N2 - The site of confluence of the artery and the portal vein in the liver still appears to be controversial. Anatomical studies suggested a presinusoidal or an intrasinusoidal confluence in the first, second or even final third of the sinusoids. The objective of this investigation was to study the problem with functional biochemical techniques. Rat livers were perfused through the hepatic artery and simultaneously either in the orthograde direction from the portal vein to the hepatic vein or in the retrograde direction from the hepatic vein to the portal vein. Arterial how was linearly dependent on arterial pressure between 70 cm H2O and 120 cm H2O at a constant portal or hepatovenous pressure of 18 cm H2O. An arterial pressure of 100 cm H2O was required for the maintenance of a homogeneous orthograde perfusion of the whole parenchyma and of a physiologic ratio of arterial to portal how of about 1:3. Glucagon was infused either through the artery or the portal vein and hepatic vein, respectively, to a submaximally effective ''calculated'' sinusoidal concentration after mixing of 0.1 nmol/L. During orthograde perfusions, arterial and portal glucagon caused the same increases in glucose output. Yet during retrograde perfusions, hepatovenous glucagon elicited metabolic alterations equal to those in orthograde perfusions, whereas arterial glucagon effected changes strongly reduced to between 10% and 50%. Arterially infused trypan blue was distributed homogeneously in the parenchyma during orthograde perfusions, whereas it reached clearly smaller areas of parenchyma during retrograde perfusions. Finally, arterially applied acridine orange was taken up by all periportal hepatocytes in the proximal half of the acinus during orthograde perfusions but only by a much smaller portion of periportal cells in the proximal third of the acinus during retrograde perfusions. These findings suggest that in rat liver, the hepatic artery and the portal vein mix before and within the first third of the sinusoids, rather than in the middle or even last third. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 067 KW - Hepatic artery KW - Portal vein KW - Confluence KW - Rat KW - Animal KW - Experimental study KW - Anatomy KW - Biochemical analysis KW - Technique KW - Perfusion KW - Exploration Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16702 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Oppermann, Martin A1 - Neuschäfer-Rube, Frank A1 - Götze, Otto A1 - Jungermann, Kurt T1 - Differential effects of human anaphylatoxin C3a on glucose output and flow in rat liver during orthograde and retrograde perfusion : the periportal scavenger cell hypothesis N2 - 1) During orthograde perfusion of rat liver human anaphylatoxin C3a caused an increase in glucose and lactate output and reduction of flow. These effects could be enhanced nearly twofold by co-infusion of the carboxypeptidase inhibitor MERGETPA, which reduced inactivation of C3a to C3adesArg. 2) During retrograde perfusion C3a caused a two- to threefold larger increase in glucose and lactate output and reduction of flow than in orthograde perfusions. These actions tended to be slightly enhanced by MERGETPA. 3) The elimination of C3a plus C3adesArg immunoreactivity during a single liver passage was around 67%, irrespective of the perfusion direction and the presence of the carboxypeptidase inhibitor MERGETPA; however, less C3adesArg and more intact C3a appeared in the perfusate in the presence of MERGETPA in orthograde and retrogade perfusions It is concluded that rat liver inactivated human anaphylatoxin C3a by conversion to C3adesArg and moreover eliminated it by an additional process. The inactivation to C3adesArg seemed to be located predominantly in the proximal periportal region of the liver sinusoid, since C3a was less effective in orthograde perfusions, when C3a first passed the proximal periportal region before reaching the predominant mass of parenchyma as its site of action, than in retrograde perfusions, when it first passed the perivenous area. These data may be evidence for a periportal scavenger mechanism, by which the liver protects itself from systemically released mediators of inflammation that interfere with the local regulation of liver metabolism and hemodynamics. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 040 Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16747 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Oppermann, Martin A1 - Muschol, Waldemar A1 - Götze, Otto A1 - Jungermann, Kurt T1 - Increase of glucose and lactate output and decrease of flow by human anaphylatoxin C3a but not C5a in perfused rat liver N2 - The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 039 KW - Hepatic glucose balance KW - Hepatic lactate balance KW - Hepatic hemodynamics KW - Complement system KW - Anaphylatoxin Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16733 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Nath, Annegret A1 - Jungermann, Kurt T1 - Increase of urate formation by stimulation of sympathetic hepatic nerves, circulating noradrenaline and glucagon inthe perfused rat liver N2 - In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 038 KW - Urate KW - Allantoin KW - Hepatic nerve KW - Catecholamine KW - Glucagon Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16728 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Hespeling, Ursula A1 - Oppermann, Martin A1 - Dieter, Peter T1 - Increase in prostanoid formation in rat liver macrophages (Kupffer cells) by human anaphylatoxin C3a N2 - Human anaphylatoxin C3a increases glycogenolysis in perfused rat liver. This action is inhibited by prostanoid synthesis inhibitors and prostanoid antagonists. Because prostanoids but not anaphylatoxin C3a can increase glycogenolysis in hepatocytes, it has been proposed that prostanoid formation in nonparenchymal cells represents an important step in the C3a-dependent increase in hepatic glycogenolysis. This study shows that (a) human anaphylatoxin C3a (0.1 to 10 mug/ml) dose-dependently increased prostaglandin D2, thromboxane B, and prostaglandin F2alpha formation in rat liver macrophages (Kupffer cells); (b) the C3a-mediated increase in prostanoid formation was maximal after 2 min and showed tachyphylaxis; and (c) the C3a-elicited prostanoid formation could be inhibited specifically by preincubation of C3a with carboxypeptidase B to remove the essential C-terminal arginine or by preincubation of C3a with Fab fragments of a neutralizing monoclonal antibody. These data support the hypothesis that the C3a-dependent activation of hepatic glycogenolysis is mediated by way of a C3a-induced prostanoid production in Kupffer cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 037 KW - lactate output KW - glucose KW - complement KW - flow KW - prostaglandin-f2-alpha Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16716 ER - TY - GEN A1 - Hespeling, Ursula A1 - Jungermann, Kurt A1 - Püschel, Gerhard Paul T1 - Feedback-inhibition of glucagon-stimulated glycogenolysis in hepatocyte/kupffer cell cocultures by glucagon-elicited prostaglandin production in kupffer cells N2 - Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 036 KW - perfused-rat-liver KW - aggregated immunoglobulin-g KW - intercellular communication KW - adenylate-cyclase KW - arachidonic-acid KW - activation KW - glucose Y1 - 1995 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16697 ER -