Cell type dependent release of nitric oxide and, or reactive nitrogen oxide species from intracellular SIN-1 [Elektronische Ressource] : effects on cellular NAD(P)H / vorgelegt von Andrea Ulrike Swintek

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• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2005
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

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   Medizinische Fakultät der Universität Duisburg-Essen    Aus dem Institut für Physiologische Chemie   Cell type-dependent release of nitric oxide and/or reactive nitrogen- oxide species from intracellular SIN-1. Effects on cellular NAD(P)H      I n a u g u r a lD i s s e r t a t i o n zur Erlangung des Doktorgrades der Medizin durch die Medizinische Fakultät der Universität Duisburg-Essen  

Vorgelegt von Andrea Ulrike Swintek aus Münster 2005

 

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    Dekan: 1.  Gutachter: 2.  Gutachter: 3.  Gutachter: Tag der mündlichen Prüfung:

 

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Univ.-Prof. Dr. rer. nat. K.-H. Jöckel Priv.-Doz. Dr. rer. nat. M. Kirsch Univ.-Prof. Dr. med. J. Fandrey Prof. Dr. rer. nat. B. Brüne, Frankfurt/M. 24. August 2005

 

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Publikation und Ergebnispräsentation: 1.) Posterwettbewerb der Universität Duisburg-Essen, Mai 2002. Titel: Erstmaliger Nachweis einer intrazellulären Stickstoffmonoxid-Freisetzung durch Molsidomin.  2.) 1. Xantener Gespräch zur Medizinisch-Biologischen Chemie zu dem Thema Molekulare Wechselwirkungen in höher organisierten Systemen, Xanten, Dezember 2002. Vortrag mit dem Titel: Erstmaliger Nachweis einer intrazellulären Stickstoffmonoxid-Freisetzung durch Molsidomin. Andrea U. Swintek, F. Petrat, M. Kirsch, H. de Groot.  3.) Publikation im Juli 2004, Biological Chemistry Titel: Cell type-dependent release of nitric oxide and/or reactive nitrogen-oxide species from intracellular SIN-1. Effects on cellular NAD(P)H. Andrea U. Swintek, Sandra Christoph, Frank Petrat, Herbert de Groot and Michael Kirsch.

 

 

List of contents: 

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1 INTRODUCTION.............................................................................................. 7

1.1 Nitric oxide............................................................................................................. 7

1.2 The sydnonimine Molsidomine.......................................................................... 9

1.3 SIN-1.................................................................................................................... 11

1.4 SIN-1C................................................................................................................. 11

1.5 Influence of pH and oxygen on decomposition of SIN-1............................. 12

1.6 Releasing compounds of SIN-1: Superoxide and nitric oxide generate peroxynitrite......................................................................................................... 12

1.7 Superoxide.......................................................................................................... 14

1.8 Aims of the study................................................................................................ 14

2 MATERIALS AND CELL CULTURE .............................................................. 16

2.1 Chemicals............................................................................................................ 16

2.2 Buffer solutions................................................................................................... 17

2.3 Cell culture of L-929 mouse fibroblasts.......................................................... 19

3 METHODS ..................................................................................................... 21

 

3.1 Determination of the uptake of SIN-1 and SIN-1C in L-929 mouse fibroblasts............................................................................................................ 21 3.1.1 Determination of cellular protein content.......................................... 22

3.2 NAD(P)H fluorescence measurement............................................................ 24 3.2.1 Recording of cellular NAD(P)H fluorescence ................................... 24 3.2.2 Determination of the subcellular distribution of NAD(P)H ................ 24

 

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3.3 Recording of the intracellular generation of reactive nitrogen-oxide species using DAF............................................................................................. 25

3.4 Determination of nitric oxide with a NO sensitive electrode........................ 27

3.5 Intracellular NO measurement with the Fluorescent Nitric Oxide Cheletropic Trap................................................................................................. 28

3.6 Cell viability......................................................................................................... 30

3.7 Statistics.............................................................................................................. 31

4 RESULTS....................................................................................................... 32

4.1 Evidence that SIN-1 penetrates L-929 cells.................................................. 32

4.2 Evidence for an intracellular production of reactive nitrogen-oxide species from SIN-1 in L929 cells.................................................................................... 34

4.3 for the generation of freely diffusing NO from intracellularlyEvidence operating SIN-1 in L-929 cells.......................................................................... 38 4.3.1 Nitric oxide sensitive electrode......................................................... 38 4.3.2 NO-scavenger F-NOCT ................................................................... 40

4.4 Evidence that the intracellular NAD(P)H level is influenced by both NO and RNOS in L-929 cells.................................................................................. 42 4.4.1 Total intracellular NAD(P)H amount ................................................. 43 4.4.2 Influence on subcellular NAD(P)H level ........................................... 44

5 DISCUSSION ................................................................................................. 47

6 CONCLUSIONS ............................................................................................. 52

7 SUMMARY ..................................................................................................... 53

8 REFERENCES............................................................................................... 54

9 APPENDIX: Abbreviations ............................................................................. 62

 

 

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ACKNOWLEDGMENTS................................................................................. 63

CURRICULUM VITAE.................................................................................... 64