Endocrinology, Vol 117, 315-322, Copyright © 1985 by Endocrine Society

ARTICLES

Protein kinase stimulation of steroidogenesis in rat luteal cell mitochondria

T Inaba and WG Wiest

The regulatory role of cAMP-dependent protein kinase in steroidogenesis was examined in luteal cell mitochondria prepared from heavily luteinized prepubertal rat ovaries. The cAMP-dependent protein kinase, localized in luteal mitochondria, comprised 5.5% of the total cellular protein kinase activity (cAMP-dependent). Intact mitochondria supported by a suitable electron-donor substrate and inhibited by isoxazole converted cholesterol to a single steroid product, pregnenolone. Neither (Bu)2 cAMP nor a crude preparation of cytosolic protein kinase stimulated pregnenolone production from cholesterol when added to intact luteal cell mitochondria; however, mitochondria treated with 10 mM CaCl2 became responsive to both (Bu)2 cAMP and protein kinase by showing increased pregnenolone production. Likewise, the addition of cytosol protein kinase to incubations of cholesterol and crude cholesterol sidechain cleavage enzyme (cytochrome P-450cscc) isolated from luteal mitochondria, also stimulated pregnenolone production. Cholesterol-poor mitochondria, depleted of endogenous sterol by prolonged preincubation, when subsequently incubated with Ca+2 plus (Bu)2 cAMP and protein kinase showed significantly increased pregnenolone production. Conversely, mitochondria with greatly increased intramitochondrial cholesterol after preincubation with 200 microM cholesterol and a cytochrome P-450cscc inhibitor (aminoglutethimide) synthesized pregnenolone in significantly higher amounts than either normal or cholesterol-poor mitochondria after removal of the aminoglutethimide block. However, addition of (Bu)2cAMP or protein kinase to Ca+2-treated cholesterol-rich mitochondria failed to additionally stimulate pregnenolone synthesis. We conclude from these observations that the mitochondrial membrane normally excludes protein kinase and (Bu)2cAMP from any stimulatory action on cholesterol side-chain cleavage. Disruption of the mitochondrial membrane by high Ca+2 concentrations eliminates this barrier and permits (Bu)2cAMP and protein kinase stimulation of the CSCC enzyme system. The mechanism of stimulation is not clear. It could involve direct action on the CSCC enzyme. Alternatively, an increase in either intramitochondrial transport or binding of cholesterol substrate to the CSCC enzyme could be facilitated by protein kinase action. Direct stimulation of the enzyme by protein kinase seems less likely, since increased enzyme activity was not observed in the presence of high concentrations of intramitochondrial cholesterol substrate.

This article has been cited by other articles:

				J. Qiao, F. C. Mei, V. L. Popov, L. A. Vergara, and X. Cheng Cell Cycle-dependent Subcellular Localization of Exchange Factor Directly Activated by cAMP J. Biol. Chem., July 12, 2002; 277(29): 26581 - 26586. [Abstract] [Full Text] [PDF]

				L. Wang, R. K. Sunahara, A. Krumins, G. Perkins, M. L. Crochiere, M. Mackey, S. Bell, M. H. Ellisman, and S. S. Taylor Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein PNAS, March 13, 2001; 98(6): 3220 - 3225. [Abstract] [Full Text] [PDF]