Skipworth, JRA; (2015) Hepatic Mitochondrial Renin-Angiotensin Systems. Doctoral thesis , UCL (University College London).

PDF James Skipworth PhD Thesis (Final)[1].pdf Download (64MB)

Abstract

Introduction: The circulating renin-angiotensin system (RAS) was originally described as a key endocrine regulator of intravascular homeostasis; however, the existence of a local (tissue) RAS has become increasingly reported in a variety of tissues including liver. RAS components have now also been detected in rat heart, brain and smooth muscle cell mitochondria as well as within intramitochondrial dense bodies of rat adrenal tissue. Further, reduced RAS levels have been associated with improved endurance performance and fatigue resistance in human skeletal muscle, suggesting that low RAS activity is associated with metabolic efficiency, potentially via RAS action upon, or within, mitochondria. However, such investigation has often relied heavily upon qualitative techniques (e.g. Western blotting, immunofluorescence and electron microscopy), which contain inherent limitations in that they completely rely upon the limited specificity of antibodies to demonstrate the existence of intra-mitochondrial RAS components. Methods: The presence of RAS components within the mitochondria of rat hepatic tissue and liver cell-lines was investigated via sub-fractionation of rat liver tissue and cell-lines, followed by Western blotting, as well as via immunofluorescence and confocal microscopy, and electron microscopy. The mitochondrial effects of stimulating or antagonizing hepatic RAS were assessed via functional fluorescence microscopy (for assessment of NADH, calcium and mitochondrial membrane potential) and measurement of oxygen consumption within live cells of a liver cell-line. Results: Western blotting, immunofluorescence and electron microscopy suggested the presence of RAS components within mitochondria; however, there was a lack of results consistency between techniques and the staining patterns were largely non-specific. Western blotting further demonstrated the presence of a prominent 55 kDa band, when immunostaining a mitochondrial fraction with (angiotensin-converting enzyme) ACE Cterminal antibody (usual size 180 kDa). This was further explored via isolation of the 55 kDa molecule and mass spectrometry to yield results consistent with non-specific staining only. Addition of RAS agonists or antagonists to live liver cell-lines demonstrated no consistent results, except at supra-physiological levels, where RAS antagonists improved oxygen consumption. Conclusions: Such data suggest that the previous descriptions of RAS components within mitochondria are likely to be secondary to methodological flaws, particularly the reliance upon single antibodies, which have subsequently been shown to have poor specificity. Thus, the effect of ang II on liver mitochondria is unlikely to be direct and any such action is likely to occur via one of several intracellular pathways, regulation of gene expression or mitochondrial biogenesis.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as JSONCSVXML

Downloads by country - last 12 months

Export as JSONCSVXML

Archive Staff Only

View Item