The in vivo effects of adrenocorticotropin and sodium restriction on the formation of different species of steroidogenic acute regulatory protein in rat adrenal

We have studied the in vivo expression of steroidogenic acute regulatory pr otein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fas-ciculata-reticularis (ZFR). An increase d signal was observed in the ZG and tend fasciculata, 5 h after ACTH inject ion; a few cells of the medulla were also positive. Immunogold electron mic roscopy showed that StAR was mainly located over mitochondria (MT). By immu no-blotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homo genates of control ZG, and their intensity was increased 5 h after ACTH tre atment but not after 1 h. Also, additional acidic species were seen 5 h aft er treatment. Other bands with basic isoelectric point were revealed betwee n 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed f our bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats . ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mob ility of some StAR species in the ZG between controls and Na+-restricted ra ts. In MT, four bands were revealed in the ZG preparations of Na+-restricte d and two bands in controls. Four bands were revealed in the ZG SNs of cont rol and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ r estriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR exp ression was inducible in the ZG and the ZF by ACTH, resulting in the format ion of many StAR acidic species; interestingly, such changes were detectabl e 5 h, but not 1 h, after ACTH administration, suggesting that steroidogene sis stimulation by StAR might occur mainly outside MT. Although less specta cular than for ACTH, Na+ restriction also affected StAR expression in the Z G but not in the ZFR, by increasing two mitochondrial and one SN species, i mplying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differentia l processing and/or changes in phosphorylation may occur in, vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relat ively small quantity of StAR, mainly located outside MT, is necessary to in crease adrenal steroidogenesis challenged either by ACTH or Na+ restriction .