St Andrews Research Repository

St Andrews University Home
View Item 
  •   St Andrews Research Repository
  • Biology (School of)
  • Biology
  • Biology Theses
  • View Item
  •   St Andrews Research Repository
  • Biology (School of)
  • Biology
  • Biology Theses
  • View Item
  •   St Andrews Research Repository
  • Biology (School of)
  • Biology
  • Biology Theses
  • View Item
  • Login
JavaScript is disabled for your browser. Some features of this site may not work without it.

The expression of Na, K-ATPase in the Madin-Darby canine kidney (MDCK) cell line

Thumbnail
View/Open
ChristopherPaulCutlerPhDThesis.pdf (51.42Mb)
Date
1991
Author
Cutler, Christopher Paul
Supervisor
Cramb, Gordon
Funder
Medical Research Council (MRC)
British Heart Foundation
Metadata
Show full item record
Altmetrics Handle Statistics
Abstract
The efficiency of a number of experimental techniques for the extraction of total RNA from various cells and tissues (including MDCK strain I cells) was assessed, and the optimal conditions for hybridisation of Na,K-ATPase isoform-specific DNA probes to this RNA were determined. The specificity of hybridisation of DNA probes for the Na,K-ATPase al, a2, a3, and beta1 isoforms was assessed using RNA isolated from rat tissues. The relative abundance of isoform mRNA's in rat kidney, brain, lung, and myocardial tissues was determined by Northern blotting. The abundance of Na,K-ATPase isoforms was also determined in the myocardial tissues of the Milan rat, a hypertensive animal model. Significant differences between the abundance of Na,K-ATPase isoform mRNA's in hypertensive rats and their age and sex matched controls were found. The relative abundance (per ng of total RNA) of al, a3, and beta1 mRNA's in left ventricle and, that of a1, and beta1 mRNA's in right ventricle were significantly decreased in hypertensive rats. The relative abundance (per mug of total RNA) of a2 and beta1 mRNA's in atria was significantly increased in hypertensive rats. These differences found in ventricles and atria were further accentuated by expression of the results per gram wet weight of tissue. The results from ventricular tissues were in contrast to those previously reported by Herrera et al. (1988) who found either increases or no change in the abundance of a1 and beta1 mRNA's in hypertensive rat aorta, skeletal muscle and left ventricle. The differences between these results may be related to the deoxy-corticosterone treatment and high salt diet of the hypertensive rat model used by Herrera et al. (1988). Na,K-ATPase isoform-specific DNA restriction endonuclease fragments were used to investigate the expression of the isoform mRNA's in MDCK strain I cells. Only a1 and beta1 mRNA's was detected on Northern blots, with no detectable a2 or a3 isoform mRNA signals being found in this cell line. [3H]-ouabain binding to cells was used, as an estimate of the cell surface expression of Na,K-ATPase. Possible factors affecting the expression of Na,K-ATPase during the normal cell growth of MDCK strain I cells were investigated. Factors such as cell seeding density, cell growth substrate and the volume of growth medium used, were all found to affect both the level and pattern of expression of Na,K-ATPase during the normal cell growth or culturing cycle. After 2 days of culture the large increases in the expression of Na,K-ATPase assayed in low density compared to high density seeded cells, were not correlated with concomitant changes in the relative abundance of Na,K-ATPase a subunit mRNA. These results indicate that the large changes in cell surface expression of Na,K- ATPase found during cell growth are probably controlled by post transcriptional processes. The effect of certain hormones or their agonists (aldosterone, deoxy-corticosterone, corticosterone, dexamethasone, and tri-iodo thyronine), on the expression of Na,K-ATPase in MDCK strain I cells was also briefly investigated. Under the conditions used, hormone treatment was not found to induce any measurable expression of a2 or a3 mRNA's. The mineralocorticoid aldosterone, and the glucocorticoid corticosterone, both produced small but significant increases in the level of Na,K-ATPase present on the cell membrane, however these increases were not correlated with similar increases in the abundance of both Na,K-ATPase a1 and beta1 mRNA's. The small size of increases in Na,K-ATPase enzyme abundance after hormone treatments and the inability of those treatments to induce consistent increases in Na,K-ATPase mRNA's further suggests that changes in the cell surface expression of Na,K-ATPase in MDCK cells is the result of regulation at a post transcriptional level.
Type
Thesis, PhD Doctor of Philosophy
Collections
  • Biology Theses
URI
http://hdl.handle.net/10023/14065

Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.

Advanced Search

Browse

All of RepositoryCommunities & CollectionsBy Issue DateNamesTitlesSubjectsClassificationTypeFunderThis CollectionBy Issue DateNamesTitlesSubjectsClassificationTypeFunder

My Account

Login

Open Access

To find out how you can benefit from open access to research, see our library web pages and Open Access blog. For open access help contact: openaccess@st-andrews.ac.uk.

Accessibility

Read our Accessibility statement.

How to submit research papers

The full text of research papers can be submitted to the repository via Pure, the University's research information system. For help see our guide: How to deposit in Pure.

Electronic thesis deposit

Help with deposit.

Repository help

For repository help contact: Digital-Repository@st-andrews.ac.uk.

Give Feedback

Cookie policy

This site may use cookies. Please see Terms and Conditions.

Usage statistics

COUNTER-compliant statistics on downloads from the repository are available from the IRUS-UK Service. Contact us for information.

© University of St Andrews Library

University of St Andrews is a charity registered in Scotland, No SC013532.

  • Facebook
  • Twitter