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Adenosinrezeptoren Auf Humanen T-Lymphozyten: Modulation Durch Subtyp-Selektive Rezeptor- Agonisten Und -Antagonisten

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Adenosinrezeptoren Auf Humanen T-Lymphozyten: Modulation Durch Subtyp-Selektive Rezeptor- Agonisten Und -Antagonisten Adenosinrezeptoren auf humanen T-Lymphozyten: Modulation durch Subtyp-selektive Rezeptor- Agonisten und -Antagonisten Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Svenja K. Lacher aus Villingen-Schwenningen Bonn 2009 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Referent: Prof. Dr. Christa E. Müller 2. Referent: Prof. Dr. Gerd Bendas 3. Referent: Prof. Dr. Gabriele König 4. Referent: Prof. Dr. Hanns Häberlein Tag der Promotion: 20. März 2009 Diese Dissertation ist auf dem Hochschulserver der ULB Bonn http://hss.ulb.uni- bonn.de/diss-online elektronisch publiziert. Die vorliegende Arbeit wurde in der Zeit von April 2005 bis Dezember 2008 am Pharma- zeutischen Institut der Rheinischen Friedrich-Wilhelms-Universität Bonn unter der Leitung von Frau Prof. Dr. Christa E. Müller durchgeführt. Mein besonderer Dank gilt Frau Prof. Dr. Christa E. Müller, die mir durch ihre Unterstüt- zung die erfolgreiche Absolvierung des Qualifizierungsjahres für Fachhochschulabsolven- ten ermöglicht hat, und mir dadurch die Möglichkeit bot, diese Arbeit mit der interessanten Themenstellung anzufertigen. Ich danke ihr für die stets freundliche Betreuung, die Dis- kussionsbereitschaft und die zahlreichen Anregungen, die maßgeblich zum Gelingen dieser Arbeit beigetragen haben. Herrn Prof. Dr. Gerd Bendas danke ich für die freundliche Übernahme des Koreferates; Frau Prof. Dr. Gabriele König und Herrn Prof. Dr. Hanns Häberlein danke ich für die Mitwirkung in meiner Promotionskommision. Ich danke der deutschen Forschungsgemeinschaft für die finanzielle Unterstützung in Form eines Stipendiums für die Heranführung an die Doktorarbeit und eines Promotionsstipen- diums im Rahmen des Graduiertenkollegs 677 „Struktur und molekulare Interaktion als Basis der Arzneimittelwirkung“. Hans-Jörg und meiner Familie Der Zweifel ist der Beginn der Wissenschaft. Wer nichts anzweifelt, prüft nichts. Wer nichts prüft, entdeckt nichts. Wer nichts entdeckt, ist blind und bleibt blind. Pierre Teilhard de Chardin (1881 – 1955) Inhaltsverzeichnis I Inhaltsverzeichnis 1 Einleitung.................................................................................................................1 1.1 Grundlagen des Immunsystems .........................................................................1 1.1.1 Lymphozyten – Effektoren der adaptiven Immunantwort........................4 1.1.2 Zytokine ....................................................................................................6 1.2 Adenosinrezeptoren............................................................................................9 1.2.1 Klassifizierung der Purinrezeptoren..........................................................9 1.2.2 Struktur G-Protein-gekoppelter Rezeptoren ...........................................11 1.2.3 Aktivierung G-Protein-gekoppelter Rezeptoren .....................................14 1.2.4 G-Protein-Kopplung der Adenosinrezeptoren ........................................15 1.2.5 Allgemeine physiologische Funktionen von Adenosinrezeptoren..........17 1.3 Adenosinrezeptoren im Immunsystem.............................................................21 1.4 Ziele der Arbeit ................................................................................................27 2 Bestimmung der Adenosinrezeptorexpression auf humanen T-Lymphozyten ................................................................................................................................31 2.1 Einleitung .........................................................................................................31 2.2 Bestimmung der Adenosinrezeptor-Transkripte durch Messung der mRNA mittels Real-time-PCR .....................................................................................34 2.3 Bestimmung der Adenosinrezeptorexpressionsmuster mittels Radioligand- Rezeptor-Bindungsstudien ...............................................................................38 2.3.1 Adenosinrezeptorexpression auf primären humanen T-Lymphozyten...39 2.3.2 Adenosinrezeptorexpression auf Jurkat T-Zellen ...................................49 2.3.3 Bestimmung der Affinitäten verschiedener Adenosinrezeptorliganden an Jurkat T-Zellen........................................................................................55 2.4 Rezeptoroligomerisierung G-Protein-gekoppelter Rezeptoren........................64 2.5 Vergleich: Real-time-PCR und Radioligand-Rezeptor-Bindungsstudien........77 2.6 Zusammenfassung und Ausblick .....................................................................81 3 Charakterisierung der Adenosinrezeptor-vermittelten Calcium-Signale in humanen T-Lymphozyten ...................................................................................85 3.1 Einleitung .........................................................................................................85 3.2 Fluorimetrische Calciummessungen an Jurkat T-Zellen..................................87 3.3 Fluorimetrische Calciummessungen an primären humanen T-Lymphozyten .92 3.4 Zusammenfassung und Ausblick .....................................................................97 4 Einfluss von Adenosinrezeptorliganden auf die Proliferation humaner T-Lymphozyten ....................................................................................................99 4.1 Einleitung .........................................................................................................99 4.1.1 Proliferation humaner Lymphozyten ......................................................99 4.1.2 Phytohaemagglutinin (PHA).................................................................101 4.1.3 Adenosin und T-Lymphozytenproliferation .........................................102 Inhaltsverzeichnis II 4.2 Einfluss von Adenosinrezeptorliganden auf die Proliferation primärer humaner T-Lymphozyten..............................................................................................106 4.2.1 Etablierung eines Assays im 96-Well-Format ......................................107 4.2.2 Etablierung eines Assays im 48-Vial-Format .......................................108 4.2.3 Überprüfung der Assayetablierung durch FACS-Messungen ..............110 4.2.4 Adenosinrezeptor-Agonisten ................................................................112 4.2.5 Adenosinrezeptor-Antagonisten............................................................116 4.2.6 Weiterführende Untersuchungen zu den Effekten der Adenosinrezeptorliganden ....................................................................121 4.2.7 Die Adenosindesaminase (ADA)..........................................................126 4.3 Einfluss von Adenosinrezeptorliganden auf die Proliferation von Jurkat T- Zellen..............................................................................................................131 4.4 Zusammenfassung und Ausblick ...................................................................134 5 Charakterisierung verschiedener Baldrianextrakte als Adenosinrezeptorliganden.................................................................................137 5.1 Einleitung .......................................................................................................137 5.1.1 [35S]GTPS-Bindungsstudien - Grundlagen .........................................143 5.2 Interaktion verschiedener hydroalkoholischer Baldrianextrakte mit A1- und A2A-Adenosinrezeptoren ................................................................................145 5.2.1 Testung der Extrakte 1-5 an A1- und A2A-Adenosinrezeptoren............145 5.2.2 Testung der Extrakte 6-8 an A1- und A2A-Adenosinrezeptoren............149 5.2.3 Testung der Extrakte 9-12 an A1-Adenosinrezeptoren .........................151 5.2.4 Zusammenfassung und Diskussion.......................................................153 5.3 Interaktion von Baldrianextrakten unterschiedlicher Polarität mit A1- und A2A- Adenosinrezeptoren........................................................................................155 5.3.1 Extraktionsmethoden zur Gewinnung der Baldrianextrakte.................155 5.3.2 Radioligand-Rezeptor-Bindungsstudien der Baldrian-Rohextrakte an A1- und A2A-Adenosinrezeptoren................................................................157 35 5.3.3 [ S]GTPS-Bindungsstudien der Baldrian-Rohextrakte an A1- Adenosinrezeptoren ..............................................................................160 5.3.4 Bioassay-geführte Fraktionierung des Extraktes KL-B-1.....................163 5.3.5 Zusammenfassung und Ausblick ..........................................................168 6 Zusammenfassung und Ausblick.......................................................................171 7 Experimenteller Teil ...........................................................................................177 7.1 Allgemeine Angaben......................................................................................177 7.1.1 Geräte und Materialien..........................................................................177 7.1.2 Chemikalien ..........................................................................................179 7.1.3 Reaktionskits.........................................................................................182 7.1.4 Radioliganden .......................................................................................182
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