Modulation of Na and acetate transport by heat shock proteins (Hsp) across the rumen epithelium of sheep [Elektronische Ressource] / vorgelegt von Imtiaz Rabbani
172 Pages
English

Modulation of Na and acetate transport by heat shock proteins (Hsp) across the rumen epithelium of sheep [Elektronische Ressource] / vorgelegt von Imtiaz Rabbani

-

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer

Description

Institute of Veterinary Physiology Faculty of Veterinary Medicine Free University of Berlin Modulation of Na and acetate transport by heat shock proteins (Hsp) across the rumen epithelium of sheep Thesis submitted for the partial fulfillment of a doctoral degree in Veterinary Medicine (Dr. med. vet.) at the Free University of Berlin submitted by Imtiaz Rabbani D.V.M, M.Sc. (Hons) Veterinarian from Lahore/Pakistan Berlin 2010 Journal Nr.: 3429 Aus dem Institut für Veterinär-Physiologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin Modulation of Na and acetate transport by heat shock proteins (Hsp) across the rumen epithelium of sheep Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin (Dr. med. vet.) an der Freien Universität Berlin vorgelegt von Imtiaz Rabbani Tierarzt aus Lahore/Pakistan Berlin 2010 Journal Nr.: 3429 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. L. Brunnberg Erster Gutachter: Univ.-Prof. Dr. H. Martens Zweiter Gutachter: Univ.-Prof. Dr. A. Richter Dritter Gutachter: Univ.-Prof. Dr. K. E. Müller Deskriptoren (nach CAB-Thesaurus): rumen, epithelium, ion transport, sodium, heat shock proteins, sheep Tag der Promotion: 05.11.

Subjects

Informations

Published by
Published 01 January 2010
Reads 16
Language English
Document size 1 MB

Institute of Veterinary Physiology
Faculty of Veterinary Medicine
Free University of Berlin




Modulation of Na and acetate transport by heat shock proteins (Hsp)
across the rumen epithelium of sheep




Thesis submitted for the partial fulfillment of a doctoral degree in
Veterinary Medicine (Dr. med. vet.) at the Free University of Berlin



submitted by
Imtiaz Rabbani
D.V.M, M.Sc. (Hons)
Veterinarian from Lahore/Pakistan
Berlin 2010
Journal Nr.: 3429 Aus dem Institut für Veterinär-Physiologie
des Fachbereichs Veterinärmedizin
der Freien Universität Berlin








Modulation of Na and acetate transport by heat shock proteins (Hsp)
across the rumen epithelium of sheep





Inaugural-Dissertation zur Erlangung des Grades eines
Doktors der Veterinärmedizin (Dr. med. vet.)
an der Freien Universität Berlin



vorgelegt von
Imtiaz Rabbani
Tierarzt aus Lahore/Pakistan
Berlin 2010
Journal Nr.: 3429
Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin
der Freien Universität Berlin


Dekan: Univ.-Prof. Dr. L. Brunnberg
Erster Gutachter: Univ.-Prof. Dr. H. Martens
Zweiter Gutachter: Univ.-Prof. Dr. A. Richter
Dritter Gutachter: Univ.-Prof. Dr. K. E. Müller


Deskriptoren (nach CAB-Thesaurus):
rumen, epithelium, ion transport, sodium, heat shock proteins, sheep


Tag der Promotion: 05.11.2010





Bibliografische Information der Deutschen Nationalbibliothek

Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über
<http://dnb.ddb.de> abrufbar.

ISBN: 978-3-86664-861-6
Zugl.: Berlin, Freie Univ., Diss., 2010
Dissertation, Freie Universität Berlin
D 188

Dieses Werk ist urheberrechtlich geschützt.
Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder
Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in
irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet,
vervielfältigt oder verbreitet werden.

Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch
ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der
Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von
jedermann benutzt werden dürfen.

This document is protected by copyright law.
No part of this document may be reproduced in any form by any means without prior written
authorization of the publisher.


Alle Rechte vorbehalten | all rights reserved
 Mensch und Buch Verlag 2010 Choriner Str. 85 - 10119 Berlin
verlag@menschundbuch.de – www.menschundbuch.de
Dedicated to
My parents for their continuous support
and unselfish love
Contents:
1 General Introduction and literature review .............................................................. 2
1.1 Introduction ................................................................................................................ 3
1.2 Review of literature .................................................................................................... 5
1.2.1 Anatomy of the rumen ................................................................................................ 5
1.2.2 Rumen fermentation ................................................................................................... 6
1.2.3 Transport mechanisms across the rumen ................................................................... 7
1.2.4 Consequences of improper fermentation and absorption ......................................... 11
1.2.5 Ruminal adaptation .................................................................................................. 16
1.2.6 Stress recovery mechanisms and pH regulation ...................................................... 18 i
1.2.7 Heat shock proteins (Hsp) ........................................................................................ 24
1.2.8 Hsp70 and acidosis 28
2 Material and Methods ................................................................................................ 33
2.1 Experimental animals ............................................................................................... 35
2.1.1 Isolation, preparation, and handling of epithelial tissues/cells .................................
2.2 Ussing chamber technique ....................................................................................... 37
2.2.1 Electrical measurements ........................................................................................... 38
2.3 Calculation of ion flux rates ..................................................................................... 40
2.3.1 Measurement of Na and acetate fluxes .................................................................... 41
2.4 Estimation of heat shock proteins (Hsp70) .............................................................. 41
2.4.1 Incubation and collection of tissues/cells ................................................................. 41
2.4.2 Western blot ............................................................................................................. 42
2.4.3 Reverse transcription with quantitative polymerase chain reaction (RT-qPCR) ..... 43
2.5 Buffer solutions ........................................................................................................ 43
2.6 Inhibitors .................................................................................................................. 44
2.6.1 Amiloride ................................................................................................................. 44
2.6.2 Cariporide (HOE 642) .............................................................................................. 44
2.6.3 S3226 ........................................................................................................................ 45
2.6.4 Cycloheximide ......................................................................................................... 45
2.6.5 Triptolide .................................................................................................................. 46
2.7 Data analysis ............................................................................................................ 46
3 Results ......................................................................................................................... 47
3.1 In vitro Induction of Hsp70 ...................................................................................... 49
i 3.1.1 Effect of temperature ................................................................................................ 49
3.1.2 Effect of pH .............................................................................................................. 60
3.1.3 Effect of SCFA concentration and mucosal pH ....................................................... 63
3.1.4 Effect of mucosal osmolarity ................................................................................... 65
3.1.5 Effect of inhibitors ................................................................................................... 66
3.2 Measurement of Na flux rates .................................................................................. 69
3.2.1 Introduction .. 69
3.2.2 Effect of NHE inhibitors .......................................................................................... 69
3.2.3 Effect of Hsp70 inhibitor 72
3.3 Measurement of acetate fluxes ................................................................................. 75
3.3.1 Introduction .............................................................................................................. 75
3.4 In vivo induction of Hsp70 ....................................................................................... 77
3.4.1 Introduction .. 77
3.4.2 Measurement of Hsp70 expression .......................................................................... 79
3.4.3 ent of Na and acetate fluxes .................................................................... 80
4 General Discussion and Conclusion 83
4.1 Induction of Hsp70 ................................................................................................... 86
4.2 Modulation of Hsp70 expression by inhibitors ........................................................ 91
4.3 Hsp and transport physiology ................................................................................... 93
4.4 Na and acetate transport ........................................................................................... 94
5 Summary ..................................................................................................................... 99
6 Zusammenfassung .................................................................................................... 103
7 References ................................................................................................................. 105
8 Appendix ................................................................................................................... 151
8.1 Buffer solutions ...................................................................................................... 151
8.2 Inhibitors ................................................................................................................ 153
8.3 Electrophysiology ................................................................................................... 154
8.4 Molecular biology .................................................................................................. 155
9 Acknowledgements ................................................................................................... 157
ii List of figures
Figure 1-1: Topographic anatomy of the forestomachs. ............................................................ 5
Figure 1-2: Scheme of pathophysiology of ruminal acidosis ................................................... 12
Figure 1-3: Diurnal variation in rumen pH .............................................................................. 17
Figure 1-4: Mechanism of activation of heat shock factor ....................................................... 25
Figure 1-5: Molecular structure of Hsp70 ................................................................................ 27
Figure 1-6: Sequence of events associated with ruminal acidosis ........................................... 31
Figure 2-1: Microscopic image of a cell fraction ..................................................................... 36
Figure 2-2: Representation of Ussing chamber technique 39
Figure 2-3: Representation of the experimental design ........................................................... 42
Figure 3-1: Effect of an increase of temperature on short circuit current ................................ 50
Figure 3-2: qPCR of three tissues obtained after 30 min of exposure to 42°C. ....................... 51
Figure 3-3: Effect of temperature on Hsp70 expression in isolated rumen epithelial cells ..... 52
Figure 3-4: Time and temperature dependent increase of Hsp70 expression .......................... 54
Figure 3-5: Effect of increasing the temperature on short circuit current ................................ 56
Figure 3-6: Effect of increasing temperature on short circuit current ...................................... 57
Figure 3-7: Effect of increasing temperature on tissue conductance ....................................... 58
Figure 3-8: Effect of different temperature on Hsp70 expression ........................................... 59
Figure 3-9: Effect of mucosal pH on I in the absence or presence of SCFA ......................... 61 sc
Figure 3-10: Hsp70 expression in rumen epithelium at various mucosal pH .......................... 63
Figure 3-11: Effect of SCFA concentration on I and G ........................................................ 64 sc t
Figure 3-12: Hsp70 expression with various mucosal concentration of SCFA ....................... 65
Figure 3-13: Inhibition of Hsp70 by cycloheximide or triptolide ............................................ 67
Figure 3-14: Changes of I after mucosal application of NHE inhibitors ............................... 71 sc
Figure 3-15: Effect of NHE inhibitors on Hsp70 expression ................................................... 72
Figure 3-16: Hsp70 expression from the tissues exposed to CHX or TRP .............................. 74
-1Figure 3-17: Effect of 1.0 mmol·l amiloride on acetate transport .......................................... 76
Figure 3-18: Correlation between rumen pH and rumen temperature ..................................... 78
Figure 3-19: In vivo expression of Hsp70 ................................................................................ 79
Figure 4-1: Plot of I and G .................................................................................................... 88 sc t
Figure 4-2: Effect of pH with or without SCFA on Hsp70 expression .................................... 89
Figure 4-3: Effect of pH and SCFA concentration on Hsp expression 90
Figure 4-4: Proposed model of pH recovery in rumen epithelium .......................................... 97 i
iii Figure 8-1: Varying expression of Hsp70 with temperature and duration ............................. 155
Figure 8-2: Hsp70 expression with different mucosal pH ..................................................... 156
iv