The objectives of this application are to elucidate the role of the peripheral-type benzodiazepine receptor (PBR) in maturation of the adrenal glands, and to determine mechanisms of regulation of this receptor. PBR is expressed in all steroidogenic cells, including gonads, adrenals, placenta, and brain (glia), where it is believed to mediate transport of cholesterol to the inner mitochondrial membrane, the site of enzymes needed to convert cholesterol to pregnenolone. Inability to transfer cholesterol to the inner membrane is associated with decreased steroidogenesis. Since this is the rate-limiting step in steroidogenesis, it is critical that this process be fully understood if new and better therapeutic approaches to disorders of adrenal pathology (e.g., adrenal insufficiency or hypertrophy) are to be developed. Studies on regulation of this important receptor in have been hindered by the lack of a normal cell or animal model in which the receptor is expressed at low levels. The neonatal rat is a model of ACTH-insensitivity, since during this period adrenocortical cells respond weakly to ACTH (10 percent of adult levels). Insensitivity to ACTH is also observed in fetuses of larger mammals, and prevents neurotoxic effects of high levels of glucocorticoids on developing brain cells. Because adrenal insufficiency is the primary cause of morbidity and mortality in premature infants (glucocorticoids are required for induction of surfactant expression in fetal lungs), the process by which the developing adrenal gland matures is of great significance. Recent findings demonstrate that neonatal rat adrenals express PBR at 10 percent the level in adult adrenals. Thus, it is proposed to characterize the complete developmental profile of PBR and other factors suggested to be important in cholesterol transport, and correlate this with changes in adrenocortical sensitivity to ACTH in vivo and in vitro. PBR expression will be determined by immunoblot, Northern blot, in situ hybridization, and ligand binding assays from fetal life through weaning. The effects of chronic treatment with ACTH or cAMP on cholesterol transport, PBR expression, and steroidogenesis in vivo and in cultured neonatal adrenal cells will be examined. The ability of transfected PBR to restore ACTH sensitivity in cultured neonatal cells will also be examined. The ontogenic appearance of PBR ligands will be determined by radioimmunoassay. These studies, therefore, will simultaneously address two important questions: What are the factors that limit steroidogenesis in immature adrenal glands, and which factors regulate expression of PBR?
