Ultrastructural Localization of Cyclic Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase after Adrenocorticotropin Stimulation in Adrenal Cortical Tumor Cells*

Abstract

To increase our knowledge of the molecular details of peptide hormone action, a specific immunogold staining procedure for the ultrastructural localization of cAMP-dependent protein kinase regulatory (RI) and catalytic (C) subunits was used in Y-1 adrenal cortical tumor cells. The Y-1 adrenal cell responds to ACTH (40 mU/ml) with a decrease in cell division and an increase in steroid production. Corresponding to the decrease in rate of cell division and the increase in steroid production, there was a 2-fold increase in both nuclear and cytoplasmic localization of the C subunit after 60-min ACTH (40 mU/ml) treatment of the culture. The amount of immunogold staining in the adrenal tumor cells after localization with antiserum of the RI subunit decreased after 60-min ACTH (40 mU/ml) stimulation. A 2-fold decrease in labeling in the cytoplasm and nucleus was observed for the RI subunit. The loss of RI subunit from the soluble fraction of the cytoplasm of the cell or an alteration of this RI subunit is suggested by this investigation. Since there was no increase in the RI subunit in the nuclear compartment, a loss of RI subunit from the cytoplasm into the nucleus seems unlikely. The observed immunogold changes in the C subunit after ACTH treatment correspond to the reported changes observed with light microscopic techniques with a fluorescein-coupled inhibitor as a probe for the localization of free C. The immunogold technique allows for the ultra-structural identification and quantification of nuclear as well as the cytoplasmic sites of cAMP-dependent protein kinase after hormonal stimulation. These results support the proposed role of protein kinase as a mediator of the ACTH response.