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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:750908 |
| Date | January 1991 |
| Creators | Cutler, Christopher Paul |
| Contributors | Cramb, Gordon |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/14065 |
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