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Title: The effect of chloride ions on steroidogenesis
Author: Ramnath, Helen Indira
ISNI:       0000 0001 3506 4926
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1997
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Both biochemical and electrophysiological studies have indicated that chloride ions may have a regulatory role in the control of steroidogenesis in rat Leydig cells (Duchatelle and Joffre, 1987; Choi and Cooke, 1990). The aim of this thesis was to elucidate the mechanisms involved using the mouse MA-10 Leydig tumour cell as a model. It was found that there is inverse relationship between extracellular chloride concentration, basal progesterone, and sub-maximally (dbcAMP) stimulated progesterone production. cAMP accumulation was also enhanced with decreasing chloride ion concentration although there was no significant effect of chloride on 125I-hCG/LHR receptor interactions at 4°C. Sub-maximal dbcAMP-stimulated progesterone production was significantly potentiated (p≤0.001) in chloride-free buffer and depended on extracellular calcium since this was decreased m calcium-free conditions (77.2±8.2%). In contrast, dbcAMP-stimulation in EGTA-treated Waymouth's medium was not as markedly decreased (38±8%). Basal progesterone was significantly increased in depolarising buffers in which potassium was increased and the sodium was decreased (p≤0.001). Titration of 0.1mM dbcAMP against decreasing chloride concentrations confirmed the specificity of the potentiation in progesterone production to decreasing chloride. Kinetic analysis showed there was a 1-2 fold increase over 6 hours for basal progesterone production in chloride-free buffer and a 15-fold increase in the presence of 0.1 mM dbcAMP. Basal and stimulated P450cscc and 3βHSD activities were unaffected by chloride-free buffer. Cycloheximide did not inhibit basal progesterone during 2 hours but after 6 hours it was significantly enhanced (p≤0.001). Cycloheximide inhibited dbcAMP sub-maximally stimulated progesterone in chloride-replete and chloride-free buffer during 2 and 6 hour incubations. A correlation was found between dbcAMP-stimulated progesterone production and the Steroidogenic Acute Regulatory (StAR) protein expression in chloride-free buffer. The StAR protein was enhanced in chloride-free buffer (4-fold). Omission of chloride had no effect on P450cscc and 3βHSD expression and there was no effect of dbcAMP on stimulated enzyme expression with or without chloride. Total protein synthesis (measured by the incorporation of 3H-methionine) was 4-fold higher in cells incubated in chloride-free buffer compared with chloride-replete buffer during stimulation with 0.1 mM dbcAMP. From the results obtained it is proposed that the enhancement of sub-maximally stimulated progesterone by omission of chloride ions occurs via the increased synthesis of the StAR protein. This may be via a general increase in cAMP-dependent protein synthesis and/or by specific enhancement of the steroidogenic effects of the StAR protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry