Die Rolle der PI-3-Kinasen in der Regulation der Sekretion von Magensäure [Elektronische Ressource] = Role of PI-3 kinases in the regulation of gastric acid secretion / vorgelegt von Anand Rotte

Die Rolle der PI-3-Kinasen in der Regulation der Sekretion von
Magensäure

Role of PI-3 kinases in the regulation of gastric acid secretion






DISSERTATION

der Fakultät für Chemie und Pharmazie
der Eberhard Karls Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften


2009


vorgelegt von
Anand Rotte
Nizamabad, India









































Tag der mündlichen Prüfung: 15.10.2009
Dekan: Prof. Dr. L. Wesemann
1. Berichterstatter: Prof. Dr. F. Lang
2. Berichterstatter: Prof. Dr. P. Ruth






To my
Father ‘Late’ Sri R Rajeshwer Rao,
Mother Srimati R Radha
and rest of the family

For all their support and encouragement
3
Table of contents
1 ACKNOWLEDGEMENTS ............................................................1
2 SUMMARY.....................................................................................3
3 ZUSAMMENFASSUNG................................................................5
4 INTRODUCTION ...........................................................................8
Significance............................................................................................................................ 8
Gastric gland (structure, location and cells involved in secretion) ...................................... 10
Parietal cells ..................................................................................................................... 10
Enterochromaffin-like cells (ECL cells) .......................................................................... 11
G cells............................................................................................................................... 12
D cells............................................................................................................................... 12
Chief cells......................................................................................................................... 12
Mucous producing cells ................................................................................................... 12
Control of secretion.............................................................................................................. 13
Neuronal ........................................................................................................................... 13
Hormonal.......................................................................................................................... 13
Secretory machinery............................................................................................................. 14
+ +H /K -ATPase .................................................................................................................. 14
+
K channels....................................................................................................................... 15
-
Cl channels ...................................................................................................................... 16
Signal transduction............................................................................................................... 17
Pathways (gastrinergic, histaminergic, cholinergic) ........................................................ 17
PI3-kinases ........................................................................................................................... 19
Physiological role............................................................................................................. 19
Rationale for the role in gastric acid secretion................................................................. 20
5 AIMS OF THE STUDIES.............................................................21
4
6 MATERIALS ................................................................................22
Equipment ............................................................................................................................ 22
Chemicals ............................................................................................................................. 22
Kits and Antibodies.............................................................................................................. 24
Animals ................................................................................................................................ 24
Diet ....................................................................................................................................... 25
7 METHODS....................................................................................26
Intracellular pH measurement .............................................................................................. 26
+
Gastric H secretion.............................................................................................................. 27
Serum gastrin........................................................................................................................ 29
Serum corticosterone............................................................................................................ 29
Western blotting ................................................................................................................... 29
In situ hybridization of SGK1 mRNA.................................................................................. 30
Immunofluorescence ............................................................................................................ 31
Quantitative real-time polymerase chain reaction measurements........................................ 32
Flow cytometric analysis (FACS) of KCNQ1 expression in the isolated parietal cells ...... 33
Statistics ............................................................................................................................... 33
8 RESULTS ......................................................................................34
i. Role of SGK1 in regulation of acid secretion ................................................................... 34
a. Role of SGK1 in regulation of basal and stimulated acid secretion............................. 34
b. Role of SGK1 in glucocorticoid induced stimulation of acid secretion....................... 36
c. Role of SGK1 in peroxisome proliferator activated receptor gamma (PPAR ) agonist γ
mediated stimulation of acid secretion............................................................................. 43
Min/+
d. SGK1 dependant stimulation of gastric acid secretion in mutant APC mice (apc )
.......................................................................................................................................... 50
ii. Role of PDK1 in regulation of acid secretion .................................................................. 58
5
9 DISCUSSION................................................................................66
i. Role of SGK1 in regulation of acid secretion ................................................................... 66
a. Role of SGK1 in regulation of basal and stimulated acid secretion............................. 66
b. Role of SGK1 in glucocorticoid stimulation of acid secretion .................................... 67
c. Role of SGK1 in peroxisome proliferator activated receptor gamma (PPAR ) agonist γ
mediated stimulation of acid secretion............................................................................. 68
Min/+
d. SGK1 dependant stimulation of gastric acid secretion in mutant apc mice (apc ). 69
ii. Role of PDK1 in regulation of acid secretion .................................................................. 71
10 ABBREVIATIONS.......................................................................73
11 REFERENCES ..............................................................................75
12 PUBLICATIONS ..........................................................................83
13 AKADEMISCHE LEHRER .........................................................84
14 LEBENSLAUF..............................................................................85
6 Acknowledgements I
1 ACKNOWLEDGEMENTS

As I stand at the threshold of earning my doctorate, I am overwhelmed when I recall all the
people who have helped me get this far. First and foremost, I would like to thank my Ph.D.
advisor, Professor Dr Florian Lang, for his constant support, guidance and inspiration.
Professor Lang is a truly remarkable advisor who gives his students lots of new ideas and
interacts closely with them. I have greatly benefited both from his excellent technical advice
and from h