Choi, Michael Shui Kuen; (1992) The involvement of calcium and chloride ions in the regulation of rat Leydig cell steroidogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cyclic AMP (cAMP) has been established as a second messenger in the stimulation of testosterone production by luteinizing hormone (LH). The aim of this thesis was to investigate the possible involvement other regulators mediating the acute stimulation of steroidogenesis in purified testis Leydig cells. The possible involvement of calcium ions, chloride ions, pH, protein kinase C (PKC) and de novo synthesis of cholesterol were investigated. Extracellular calcium was found to be essential for both maximum steroidogenesis and cAMP production. However, LH-stimulated increase in intracellular calcium could not be reproducibly demonstrated in cells loaded with fluorescent calcium indicator Fura 2. The involvement of calmodulin in testosterone production was investigated using the calmodulin inhibitor, calmidazolium. This compound inhibited cAMP production stimulated by LH, forskolin and cholera toxin, with ID50 of 2μM. It had both a stimulatory and inhibitory effect on steroidogenesis. The stimulatory effect was an unexpected finding; it was independent of cAMP, calcium and protein synthesis, and was similar in magnitude to stimulation obtained with maximum levels of LH or dibutyryl cyclic AMP (dbcAMP). It also stimulated steroidogenesis in rat adrenocortical cells and mouse Leydig cells. Using cells loaded with the fluorescent pH indicator BCECF, the intracellular pH was found to be pH 7.2, and the presence of the Na+/H+ exchanger was demonstrated. Testosterone production was found to be relatively insensitive to fluctuations in intracellular pH. It has been established that rat Leydig cells possess outward rectifying chloride channels on their plasma membrane. The replacement of extracellular chloride with gluconate salts caused a potentiation of the sub-maximal testosterone production in response to low concentrations of LH and dbcAMP. This potentiating effect of chloride replacement was also found in dbcAMP-stimulated pregnenolone production in rat adrenocortical cells. These results suggest that the efflux of chloride ions may play an important role in steroidogenesis. The chloride channel inhibitors, SITS and DIDS, inhibited both LH-stimulated cAMP and testosterone production, but had no effect on dbcAMP stimulated steroidogenesis. The precise role of SITS- and DIDS-sensitive chloride channels remains unclear. Both basal and stimulated testosterone production were stimulated two-fold when PKC activity was downregulated through preincubation with phorbol ester, phorbol 12- myristate, 13-acetate (PMA). Preincubation with an inactive phorbol ester, phorbol 12,13-dideconoate (PDD), had no effect on testosterone production. The site of this potentiation was prior to cholesterol-side-chain-cleavage. Testosterone production in Leydig cells appears to be tonically inhibited by the action PKC. Pertussis toxin-sensitive G-protein(s) has previously been found in Leydig cells. In the present studies, pertussis toxin was found to inhibit both LH- and dbcAMP-stimulated testosterone production. This suggests the involvement of pertussis toxin- sensitive G-proteins in the regulation of testosterone production. The supply of cholesterol for testosterone production in rat Leydig cells is derived from de novo synthesis. It was found that de novo synthesis of cholesterol was not required for acute testosterone production. This indicates the existence of a large pool of steroidogenic cholesterol in rat Leydig cells.

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