Developmental expression of the peripheral-type benzodiazepine receptor and the advent of steroidogenesis in rat adrenal glands.

Although the precise mechanism whereby cholesterol is transported across the outer mitochondrial membrane is uncertain, a multimeric receptor complex termed the peripheral-type benzodiazepine receptor (PBR) appears essential for this process. We therefore predicted that adrenal cells at different developmental stages would express PBR coincidentally with the advent of steroidogenesis. Adrenals of neonatal rats demonstrate greatly reduced sensitivity to ACTH that gradually increases after the first 2 weeks of life. Thus, neonates have lower circulating corticosterone levels following exposure to stress. We examined mitochondrial PBR ligand binding activity, immunoreactive (ir) PBR content, and adrenal sensitivity to ACTH in vivo and in vitro. Ontogeny of both mitochondrial PBR ligand binding capacity and irPBR directly paralleled that of ACTH-inducible steroidogenesis in isolated rat adrenal cells and in rats injected with ACTH. In addition, neonatal PBR had approximately 2-fold higher affinity for PK11195, a synthetic ligand that binds with high affinity to PBR. No correlation was observed during neonatal life between ir-steroidogenic acute regulatory (StAR) protein content and steroidogenesis. These results are consistent with the hypothesis that PBR is an absolute prerequisite for adrenocortical steroidogenesis, and suggest that the stress hyporesponsive period of neonatal rats may result from decreased PBR expression. In addition, the higher affinity of neonatal PBR and the relatively high basal expression of StAR protein in neonatal adrenals may partly explain the high constitutive steroidogenesis characteristic of neonatal rat adrenal cells.

Differential sensitivity to ACTH, but not stress, in two sources of outbred Sprague-Dawley rats.

Adrenocorticotropic hormone (ACTH) is the major regulator of adrenocortical steroidogenesis in mammals. By comparing the sensitivity to ACTH of isolated adrenocortical cells from two sources of the same strain (Sprague-Dawley, SD) of outbred rats, we have identified a source of rat with low sensitivity to ACTH in vitro. Cells isolated from Holtzman SD rats had a high sensitivity to ACTH (minimal effective concentration 50 pg/ml), whereas Taconic SD rats had a low sensitivity (minimal effective concentration 250 pg/ml; maximal steroidogenesis < 50% of Holtzman cells). The responsiveness to analogues of cyclic adenosine monophosphate and cholesterol was also significantly lower in Taconic SD rats. Taconic adrenals were smaller, had significantly more mitochondria per cell, but approximately 20% less total lipid droplet volume per cell. There was no difference in latency to ACTH in vitro; however, steroidogenesis plateaued in Taconic cells after 25 min, while Holtzman cells secreted corticosterone almost linearly for at least 120 min. By contrast, the cyclic adenosine monophosphate secretion increased at the same rate for at least 120 min in cells from both sources. There were no differences between cells from the two sources in immunoreactive steroidogenic enzyme content. In vivo, the magnitude of the ACTH and corticosterone responses to two types of stress were similar in both sources. The thymus glands of Holtzman rats were significantly larger than those of Taconic rats. It is concluded that: (1) reduced sensitivity to ACTH in vitro in Taconic SD rats results from differences in the later stages of the steroidogenic pathway; (2) factors in addition to ACTH are required for maximal steroidogenesis in Taconic SD rats: (3) a comparison of the steroidogenic pathways in adrenal cells from these two sources of outbred rats should be useful in further delineating the relative importance of putative intracellular signalling mechanisms involved in initiation and maintenance of steroidogenesis, and (4) these data suggest that different sources of the same strain of rats sufficiently diverge over time to become separate strains ('substrains'). Overreliance on a single source of laboratory rodent may obscure natural variability in endocrine responses to stress and provide a misleading indication of homogeneity of responses.

In vivo studies on the role of the peripheral benzodiazepine receptor (PBR) in steroidogenesis.

In various steroidogenic cell models, mitochondrial preparations and submitochondrial fractions, the expression of the mitochondrial 18 kDa peripheral-type benzodiazepine receptor (PBR) protein confers the ability to take up and release, upon ligand activation, cholesterol. Thus, cholesterol becomes available to P450scc on the inner mitochondrial membrane. These in vitro studies were validated by in vivo experiments. Treatment of rats with ginkgolide B (GKB), specifically reduced the ligand binding capacity, protein, and mRNA expression of the adrenocortical PBR and circulating glucocorticoid levels. Treatment with GKB also resulted in inhibition of PBR protein synthesis and corticosterone production by isolated adrenocortical cells in response to ACTH. The ontogeny of both PBR binding capacity and protein directly paralleled that of ACTH-inducible steroidogenesis in rat adrenal cells and in rats injected with ACTH. In addition, the previously described suppression of luteal progesterone synthesis in the pregnant rat by continuous in vivo administration of a gonadotropin-releasing hormone agonist may be due to decreased luteal PBR ligand binding and mRNA. These results suggest that (i) PBR is an absolute prerequisite for adrenocortical and luteal steroidogenesis, (ii) regulation of adrenal PBR expression may be used as a tool to control circulating glucocorticoid levels and (iii) the stress hypo-responsive period of neonatal rats may result from decreased adrenal cortical PBR expression.