Georgiou M, Perkins L M, Payne A H
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0278.
Endocrinology. 1987 Oct;121(4):1390-9. doi: 10.1210/endo-121-4-1390.
Treatment of primary cultures of rat Leydig cells with 1 mM 8-bromo-cAMP for 2 days at ambient oxygen tension (19%) caused a 59% decrease in mitochondrial cholesterol side-chain cleavage (P-450scc) activity. This decrease was completely prevented when the oxygen tension was reduced to 1% O2 or when steroid synthesis was inhibited by aminoglutethimide. When the endogenous concentration of pregnenolone was increased by inhibiting its further metabolism, P-450scc activity was reduced by 80% in unstimulated cultures and was completely eliminated in cAMP-treated cultures. These losses were prevented when cells were maintained at 1% O2. The amount of immunoreactive P-450scc was also decreased by treatments that reduced P-450scc activity. Stimulation with cAMP also lowered microsomal C17-20 lyase activity by an oxygen-mediated, steroid synthesis-dependent mechanism. Treatment of cultures with testosterone caused a similar oxygen tension-sensitive decrease in C17-20 lyase activity. These results suggest that the enhanced loss of mitochondrial and microsomal cytochrome P-450 activities in cAMP-treated cultures is caused by the increased production of pregnenolone and testosterone, respectively, which generate reactive damaging species derived from reduced dioxygen. The increased catalytic turnover of these P-450 enzymes may also contribute to their damage. Although P-450 activities were preserved at 1% O2, the ability of cAMP-treated cells to synthesize testosterone in response to subsequent cAMP stimulation was still reduced. If, however, 25-hydroxycholesterol was supplied to these cells the decrease in testosterone-producing capacity was prevented, which demonstrates that the reduced steroidogenic capacity of cAMP-treated Leydig cells is caused, primarily, by the reduced availability of endogenous cholesterol.
在环境氧张力（19%）下，用1 mM 8-溴环磷腺苷（8-bromo-cAMP）处理大鼠睾丸间质细胞原代培养物2天，导致线粒体胆固醇侧链裂解酶（P-450scc）活性降低59%（P<0.05）。当氧张力降至1% O₂ 或用氨鲁米特抑制类固醇合成时，这种降低可完全被阻止。当通过抑制孕烯醇酮的进一步代谢来提高其内源性浓度时，未受刺激的培养物中P-450scc活性降低80%，而在经cAMP处理的培养物中则完全消除。当细胞维持在1% O₂ 时，这些损失可被阻止。降低P-450scc活性的处理也会减少免疫反应性P-450scc的量。用cAMP刺激也会通过氧介导的、类固醇合成依赖性机制降低微粒体C17-20裂解酶活性。用睾酮处理培养物会导致C17-20裂解酶活性出现类似的氧张力敏感性降低。这些结果表明，在经cAMP处理的培养物中，线粒体和微粒体细胞色素P-450活性的增强损失分别是由孕烯醇酮和睾酮产量增加所致，它们产生了源自还原型双加氧的反应性损伤物质。这些P-450酶催化周转率的增加也可能导致其损伤。尽管在1% O₂ 时P-450活性得以保留，但经cAMP处理的细胞对随后cAMP刺激合成睾酮的能力仍降低。然而，如果向这些细胞提供25-羟胆固醇，则可防止睾酮生成能力的降低，这表明经cAMP处理的睾丸间质细胞类固醇生成能力降低主要是由内源性胆固醇可用性降低所致。
