title: The effects of different culture media, glucose, pyridine nucleotides and adenosine on the activity of 11beta-hydroxysteroid dehydrogenase in rat Leydig cells.
abstract: 11Beta-hydroxysteroid dehydrogenase (11betaHSD) reversibly converts glucocorticoids into inert 11-ketosteroids. The direction of the reaction has been found to vary with the cell type and sub-cellular preparation used. We have investigated if the directionality of 11betaHSD can be influenced by the nature of the culture medium and compounds added during incubation of rat testis Leydig cells. We found that when the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) that the dehydrogenase (11betaDH) activity was higher than the reductase (11KSR) activity (11betaDH:11KSR ratio approximately 2:1). When glucose was omitted from the DMEM a higher 11betaDH:11KSR ratio (approximately 33:1) was obtained. However, when the cells were cultured in a combination of DMEM/Ham's F12 (1:1, v/v), a ninefold increase in 11KSR activity was obtained whereas 11betaDH activity was inhibited by 64% compared with cells incubated in DMEM alone. Consequently, the predominant activity changed from a dehydrogenase to a reductase (11betaDH:11KSR ratio 1:15). Addition of the individual components of the Ham's F12 medium to DMEM showed that only pyruvate and/or the amino acids were able to mimic the effects of DMEM/Ham's F12. Similar differential effects were found when NAD+, NADH or adenosine were added to the Leydig cells incubated in DMEM (three to fivefold increases and 20-50% decreases in 11KSR and 11betaDH activities, respectively). In contrast, NADP+ was found to increase 11betaDH activity (up to threefold) but NADPH had no effect on 11KSR activity. Cells incubated with DMEM/Ham's F12, NAD+, NADP+ and adenosine were found to have higher ATP levels (four to sixfold) than those incubated in DMEM alone. These results illustrate that the relative 11betaDH and 11KSR activities of 11betaHSD in Leydig cells are markedly and differentially altered by the nature of the incubation medium and compounds added.
authors.affiliation: Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School of University College London, Royal Free Campus, UK.
authors.forename: S E
authors.initials: SE
authors.lastname: Ferguson
authors.forename: Z
authors.initials: Z
authors.lastname: Pallikaros
authors.forename: A E
authors.initials: AE
authors.lastname: Michael
authors.forename: B A
authors.initials: BA
authors.lastname: Cooke
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D000241:Adenosine / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D005947:Glucose / Q000494:pharmacology*; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D009243:NAD / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D003470:Culture Media; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D006913:Hydroxysteroid Dehydrogenases; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D005947:Glucose; D000241:Adenosine