Robert C. Pedersen

Steroidogenesis activator polypeptide (SAP) in the rat ovary and testi

A steroidogenesis activator polypeptide (SAP) has previously been identified in the rat adrenal cortex (Pedersen and Brownie, Proc. natn. Acad. Sci. U.S.A. 80 (1983) 1882-1886). This factor apparently facilitates the association of mitochondrial cholesterol with the cholesterol side-chain cleavage cytochrome P-450, a reaction which is generally regarded as rate-controlling in the steroid biosynthetic pathway. The same preparative techniques have now been applied in a search for this material in other rat tissues. Among those investigated, the ovary and testis demonstrate significant concentrations of a factor which is biologically and chromatographically similar to adrenal SAP. In the immature ovary the activator becomes manifest after priming with PMSG and rises dramatically during hCG-stimulated luteinization, an increase which can be blunted with cycloheximide. In the adult rat testis it is increased acutely by treatment with hCG or dibutyryl cAMP and is diminished in response to hypophysectomy or cycloheximide. At approximately equivalent concentrations (10(-7) M), preparations of the activator from the adrenal cortex, the testis, and the superovulated ovary each enhance the activity of cholesterol side-chain cleavage in adrenocortical mitochondria by 5- to 6-fold over basal controls. We conclude that steroidogenic organs share a similar or identical intracellular modulator of cholesterol----pregnenolone conversion which is under pituitary control.

Steroidogenesis activator polypeptide (SAP) in the guinea pig adrenal cortex.

Steroidogenesis activator polypeptide (SAP), a cytosolic stimulator of cholesterol side-chain cleavage (cholesterol SCC) previously characterized in the rat, was isolated from guinea pig adrenal cortex. This factor exhibited behavior on reverse-phase high-performance liquid chromatography (HPLC) that was indistinguishable from authentic SAP and crossreacted fully in a SAP radioimmunoassay. In dexamethasone-suppressed guinea pigs neither the concentrations of immunoreactive adrenal SAP nor the levels of cholesterol SCC activity were significantly different between the outer zones (zonae glomerulosa and fasciculata) and the inner zone (zona reticularis). However, at 10 min after treatment of dexamethasone-suppressed animals with ACTH1-24, the outer zone content of SAP was increased 42-fold over unstimulated controls whereas inner zone SAP was elevated only 4-fold. At the same time, cholesterol SCC activity was increased 2-fold in the outer zones but unchanged in the inner zone. In addition to SAP itself, a crossreacting 82 kDa protein (p82)--similar to the putative SAP precursor identified in the rat--was detected on two-dimensional immunoblots of guinea pig whole adrenal homogenate. There were no significant differences in the protein concentrations of p82 or of cytochrome P-450scc between zones, either with or without ACTH treatment. We conclude that the widely reported contrast in corticosteroidogenic potential between the zona fasciculata and the zona reticularis of the guinea pig may reflect a differential capacity to generate SAP, and thus activate cholesterol SCC, in response to ACTH.

Failure of ACTH to mimic the stress-induced activation of rat adrenocortical cholesterol ester hydrolase in vivo

Abstract Rat adrenocortical cholesterol ester hydrolase activity does not respond to the circadian rhythm of the pituitary-adrenal axis under quiescent conditions. However, stress activates this enzyme, producing a greater increase in activity at the high point of the cycle than at the low point. This stress-induced effect requires the presence of the pituitary. It is not mimicked by administration of exogenous porcine ACTH to either hypophysectomized or dexamethasone-suppressed rats, but homogenates of whole rat pituitary tissue do possess substantial stimulatory capacity.

Steroidogenesis Activator Polypeptide May be a Product of Glucose Regulated Protein 78 (GRP78)

Cholesterol side-chain cleavage is sensitive to antibiotic inhibitors of protein synthesis, suggesting that a labile protein may play a regulatory role in this process. We have previously characterized such a factor--steroidogenesis activator polypeptide (SAP). Given the low molecular weight of SAP (Mr 3215), a SAP precursor has been sought. Using immunoblotting techniques with two polyclonal antisera directed against portions of the SAP sequence, a single protein of apparent Mr 82,000 (p82) can be detected in rat adrenocortical tissue. Our data suggest that adrenal p82 is most likely the widely-distributed minor heat shock protein, glucose regulated protein 78 (GRP78). The two proteins share biochemical attributes, including pI (5.2) and ATP affinity, and the reported amino acid sequences for SAP and for the carboxyl-terminal end of GRP78 are nearly identical. We propose that SAP is cleaved from GRP78--or a cognate protein--and that this proteolysis is regulated in a manner characteristic of steroidogenic tissues.

Gamma3-melanotropin promotes mitochondrial cholesterol accumulation in the rat adrenal cortex

Abstract Gamma 3-melanotropin (γ3-MSH) facilitates a rapid, dose-dependent, and cycloheximide-insensitive increase in the concentration of mitochondrial free cholesterol in the adrenals of hypophysectomized rats. Physiological concentrations of various synthetic and native preparations of γ3-MSH are potent, while γ-MSH is not. This cholesterol accumulation coincides with the activation of cholesteryl ester hydrolysis by γ3-MSH, while the rates of cholesterol esterification and of mitochondrial cholesterol side-chain cleavage are unaffected. Conversely, ACTH inhibits cholesterol esterification. Therefore, γ3-MSH and ACTH together may coordinate a substantial shift in the set-point of cholesteryl ester ag cholesterol cycling toward the right. Because ACTH also activates cholesterol side-chain cleavage, this coordinate effect on the flux of cholesterol substrate is manifest as a potentiation of Corticosteroidogenesis by γ3-MSH. These data extend our previous studies demonstrating that pro-y-MSH polypeptides have an endocrine influence on the rat adrenal cortex.

High density lipoprotein influences the response of rat adrenocortical cells to λ3-melanotropin

Abstract Gamma 3 -melanotropin (γ 3 -MSH) exhibits a marked dose-dependent synergism with ACTH 1–24 on corticosterone production by cells isolated from the inner zones of the rat adrenal cortex. This phenomenon is demonstrated to best advantage when donor animals are killed after stress or pretreatment with ACTH. If adrenal cells are prepared from quiescent or hypophysectomized animals, the inclusion of high density lipoprotein (HDL) in the incubation medium is required for a significant γ 3 -MSH response. Dibutyryl cAMP can successfully substitute for ACTH 1–24 in these incubations but rat low density lipoprotein does not reproduce the HDL effect. These data are consistent with our in vivo studies demonstrating that γ 3 -MSH potentiation is a product of increased cholesterol mobilization within the adrenal cortex and suggest that in the rat, a significant source of the cholesteryl ester pool which is responsive to γ 3 -MSH may derive from circulating HDL.