CHOLESTEROL TRAFFICKING IN STEROIDOGENIC CELLS - REVERSIBLE CYCLOHEXIMIDE-DEPENDENT ACCUMULATION OF CHOLESTEROL IN A PRE-STEROIDOGENIC POOL

Peptide hormones activate steroid hormone biosynthesis in responsive t issues by stimulating the delivery of cholesterol to a steroidogenic p ool, thought to be located in the inner mitochondrial membrane. At thi s site, it is metabolized to pregnenolone, the precursor of the steroi d hormones, by side-chain-cleaving cytochrome P-450 (cytochrome P-450s cc). In the presence of aminoglutethimide (an inhibitor of cytochrome P-450scc) and an activating stimulus, cholesterol accumulates in the s teroidogenic pool, and increased pregnenolone generation is observed u pon removal of the inhibitor. Using Y-1 adrenocortical cells and MA-10 Leydig tumor cells, we now provide evidence for a distinct, functiona lly relevant cholesterol pool which precedes the steroidogenic pool, w hich we designate the pre-steroidogenic pool. This pool was defined by activating the cells with 8-bromo-adenosine 3',5'-cyclic monophosphor ic acid in the presence of cycloheximide, an inhibitor of steriodogene sis. Following a wash procedure, which removed 8-bromo-adenosine 3',5' -cyclic monophosphoric acid and cycloheximide, augmented pregnenolone synthesis was observed. Unlike synthesis from the steroidogenic pool, pregnenolone formation from pre-steroidogenic pool in Y-1 cells indica tes that this pool is somewhat smaller than the steroidogenic pool. Th e results support a cholesterol-trafficking model in which cycloheximi de-sensitive transport from the pre-steroidogenic pool to the steroido genic pool precedes metabolism, and is regulated by cAMP.