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Evidence that hydrogen peroxide blocks hormone-sensitive cholesterol transport into mitochondria of rat luteal cells
1Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06510.
Abstract:
In luteal and granulosa cells, hydrogen peroxide abruptly inhibits activation of adenylate cyclase by receptor-bound gonadotropin and blocks steroidogenesis. In the present studies a post-cAMP site of peroxide action on inhibition of steroidogenesis was investigated. Steroidogenesis, stimulated by dibutyryl or 8-bromo-cAMP, was inhibited by hydrogen peroxide. Yet, cAMP-dependent protein kinase activation in cytosol or intact cells was unaffected by peroxide treatment. Hydrogen peroxide also did not inhibit the activity of cholesterol esterase and acyl coenzyme-A:acyltransferase. Progesterone synthesis was maximally increased 5- to 50-fold with 25- and 22-hydroxycholesterol, respectively. Unlike that seen with cAMP analogs and LH, however, progestin synthesis stimulated by these cell- and mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide. Treatment of animals with amino-glutethimide produces a marked accumulation of steroidogenic cholesterol substrate and a large increase in hormone-independent steroidogenesis in subsequently isolated and washed luteal tissue. In this paradigm, hydrogen peroxide did not inhibit elevated basal progesterone synthesis in luteal cells produced by in vivo aminoglutethimide treatment, yet LH-stimulated steroidogenesis was blocked. However, treatment of luteal cells with hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, an effect partially reversed by the addition of luteal cell cytosol. In summary, while peroxide inhibited cAMP-dependent steroidogenesis, it did not appear to inhibit protein kinase activation or mobilization of cholesterol from intracellular esterified stores. Although peroxide inhibited pregnenolone synthesis, it had no effect on steroidogenesis when substrate was made available by either addition of cholesterol analogs or prior treatment with aminoglutethimide in vivo. We conclude, therefore, that hydrogen peroxide inhibits steroidogenesis by blocking intracellular transport of cholesterol to mitochondria or translocation of cholesterol across the outer mitochondrial membrane.
Insights
Hydrogen peroxide inhibits steroidogenesis by blocking cholesterol transport to mitochondria. This occurs after cAMP activation and affects pregnenolone synthesis, but not when cholesterol is readily available.
Area of Science:
- Cellular Biology
- Endocrinology
- Biochemistry
Background:
- Hydrogen peroxide is known to inhibit steroidogenesis in luteal and granulosa cells by blocking adenylate cyclase activation.
- The precise mechanism of peroxide's inhibitory action on steroidogenesis, particularly at a post-cAMP level, requires further investigation.
Purpose of the Study:
- To investigate the post-cAMP site of hydrogen peroxide action in the inhibition of steroidogenesis.
- To elucidate the specific molecular targets and pathways affected by hydrogen peroxide during steroid hormone production.
Main Methods:
- Steroidogenesis was stimulated using cAMP analogs (dibutyryl or 8-bromo-cAMP) and cholesterol analogs (25- and 22-hydroxycholesterol).
- Effects of hydrogen peroxide on cAMP-dependent protein kinase activation, cholesterol esterase, and acyl-CoA:acyltransferase activities were assessed.
- Steroidogenesis was also examined in luteal cells from animals treated with aminoglutethimide and in isolated mitochondria.
Main Results:
- Hydrogen peroxide inhibited steroidogenesis stimulated by cAMP analogs but did not affect cAMP-dependent protein kinase activation or key enzymes in cholesterol mobilization.
- Progestin synthesis stimulated by cell- and mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide.
- Hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, suggesting a role in cholesterol transport to mitochondria.
Conclusions:
- Hydrogen peroxide inhibits steroidogenesis at a post-cAMP level, likely by interfering with intracellular cholesterol transport to mitochondria or translocation across the outer mitochondrial membrane.
- The inhibitory effect is substrate-dependent, as peroxide does not block steroidogenesis when cholesterol is readily available via analogs or in vivo aminoglutethimide treatment.