Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland.

Abstract

Rat adrenal mitochondria accumulated cholesterol during ether stress in vivo when side-chain cleavage was inhibited by aminoglutethimide (control = 14.6 vs. aminoglutethimide = 26.5 micrograms of cholesterol per mg of protein). This accumulation was insensitive to simultaneous administration of cycloheximide (24.2 micrograms/mg), but side chain cleavage in the mitochondria was greatly decreased. Outer and inner mitochondrial membrane fractions were separated by discontinuous Ficoll gradient centrifugation. Quantitation of marker enzymes for inner, outer, and microsomal enzymes indicated that outer membranes contained less than 5% inner membranes. The inner membrane fraction contained less than 7% outer membrane and included 90% of mitochondrial cytochrome P-450. Electron microscopy revealed outer membranes as circular intact ghosts, whereas inner membranes were largely intact and retained vesicular structure typical of intact adrenal cortex mitochondria. Administration of aminoglutethimide effected a 2-fold increase in inner membrane cholesterol (9.4 vs. 20.1 micrograms/mg) but simultaneous administration of cycloheximide completely blocked this increase (10.9 micrograms/mg). We conclude that: (i) in the presence of aminoglutethimide, stress stimulates accumulation of cholesterol in the inner membrane of adrenal mitochondria; and (ii) transfer of cholesterol from outer to inner membranes requires a cycloheximide-sensitive agent.

Impact of Aging on Steroid Hormone Biosynthesis and Secretion

“…The tropic hormones, ACTH and gonadotropins (LH, FSH) acutely stimulate this process by facilitating the availability of cytosolic free cholesterol and its transport to and accumulation in the inner mitochondrial CYP11A1 sites. When steroidogenic cells are stimu-lated with tropic hormones in the presence of protein synthesis inhibitors such as cycloheximide, cholesterol accumulates in the outer mitochondrial membrane and is not transferred to the inner mitochondrial membrane [54][55][56][57]. Therefore, it has been proposed that a labile protein is required for cholesterol delivery to the inner mitochondrial membrane microdomains containing CYP11A1 [26,53,59].…”

“…The second critical step is the delivery of the cholesterol substrate to inner mitochondrial membrane sites of CYP11A1 [53,56,57]. As noted above, this step is considered rate-limiting because hydrophobic cholesterol cannot rapidly diffuse through the aqueous intermembrane space of the mitochondria to support acute steroid synthesis and requires the participation of a de novo synthesized labile protein [61][62][63][64][65].…”

Regulation of acute cortisol synthesis by cAMP-dependent protein kinase and protein kinase C in a teleost species, the rainbow trout (Oncorhynchus mykiss)

“…Following the finding of the implication of both kinases upstream of pregnenolone formation, it was important to assess whether PKA and PKC exert their regulatory roles on the mitochondrial cholesterol translocation, believed to be the rate limiting step in the acute response (Privalle et al 1983, Stocco & Clark 1996, Kallen et al 1998). An exogenous cholesterol substrate, 25-OH-cholesterol, which has the capacity to diffuse across the mitochondrial membranes (Toaff et al 1982) was therefore used to stimulate acute cortisol synthesis while bypassing the translocation step.…”

The 5′‐flanking region of the human P‐450(SCC) gene shows responsiveness to CAMP‐dependent regulation in a transient gene‐expression system of Y‐1 adrenal tumor cells

“…ACTH is known to regulate the steroidogenesis in two ways, exhibiting a short-term (acute) and a long-term (chronic) effect. The short-term action has been attributed to an increased utilization of cholesterol mediated by increased binding of this substrate to P-45O(SCC) [20,211, while the long-term effect has been suggested to , which contained a part of the first exon and its 5'-flanking sequence, was labeled at the 5' cnd with 32P. The anticoding strand of the fragment, which was separated in 8% polyacrylamide gel electrophoresis, was hybridized with the total RNA (5 pg) from human adrenal cortex in 20 11 at 46°C for 2.5 h and the reaction mixture was treated with S1 nuclease as described [14].…”