Both acute and chronic steroidogenesis is regulated by adrenocorticotropin (ACTH), a principal regulator of the adrenal cortex. A number of studies have demonstrated that Steroidogenic Acute Regulatory (StAR) protein plays a crucial role in facilitating cholesterol transfer from the outer to the inner mitochondrial membrane where the first step of cholesterol conversion to steroid hormones occurs. This work has evaluated the relationships between the expression of StAR protein and signalling pathways of ACTH-induced steroidogenesis in primary cultures of bovine adrenal zona fasciculata (ZF) cells. A novel sheep anti-bovine peptide StAR polyclonal antibody has been characterised and optimised for Western immunoblotting. A newly formulated protocol based on enhanced chemiluminescence methodology provided a linear, reproducible and sensitive approach to detect and quantify StAR protein by molecular imaging analysis. The expression level of StAR protein after ACTH treatment of adrenal ZF cells showed that levels of StAR were insensitive to ACTH in freshly isolated cells due to the high initial levels of the protein. The cell responsiveness to ACTH was remarkable, however, after the basal levels of StAR protein diminished to relatively lower levels after 2 days of culture. Concentration-response curves demonstrated that, in general, increasing concentrations of ACTH resulted in increasing in cortisol output in parallel with increases of cyclic adenosine 3', 5'-monophsopathe (cAMP) production; the maximal effects of ACTH on cortisol and cAMP levels exhibited with 10<sup>-8</sup> M ACTH treatment after both 1 and 6 hr. Marked changes in StAR protein occurred at 6 hr. attaining a maximal level with 10<sup>-8</sup> M ACTH. Interestedly, at 1 and 6 hr the elevation of cortisol levels were significantly altered compared to the basal levels at 10<sup>-12</sup> M ACTH without any notable increase in cAMP. The time courses for ACTH treatment showed that at10<sup>-8</sup> M ACTH (a supraphysiological concentration) there was a strong correlation between cortisol and StAR protein induction, suggesting that newly synthesised StAR protein may be a major mediator for steroidogenesis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663448 |
| Date | January 2001 |
| Creators | Wang, Hui |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/23243 |
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