Adrenal Cells in Tissue Culture. X. On the Mechanism of the Stimulation of Steroidogenesis by Cyclic Cytidine Monophosphates12

Abstract

In monolayer cultures of mouse adrenal cortex cells, cyclic cytidine monophosphate (cyclic CMP) and cyclic AMP are comparably steroidogenic. Cyclic CMP induces the same morphological changes in the cells as cyclic AMP and ACTH. The stimulatory effects of cyclic CMP are not associated with increases in intracellular cyclic AMP. Cyclic AMP levels were measured by the binding assay of Gilman and by measuring the levels of ¹⁴C-cyclic AMP in cells previously labeled with ¹⁴C-(ul)-adenine. ACTH increased cyclic AMP at least 4-fold by both these methods. Cyclic CMP is capable of displacing cyclic AMP from cyclic AMP-binding proteins in the cytosol of adrenal cell extracts. A 100-fold higher concentration of cyclic CMP than cyclic AMP is required. Cyclic GMP, which is not steroidogenic in these cells binds with the same affinity as cyclic CMP. Protein kinase activity is also increased by cyclic CMP and cyclic GMP at 100-fold higher concentrations than cyclic AMP. Stimulation of protein kinase is greater with both cyclic CMP and cyclic GMP than with cyclic AMP at maximally stimulatory levels of the nucleotides. Extracts of cells stimulated with cyclic CMP contained a significant amount of contaminating cyclic CMP, whereas extracts of cells treated with cyclic AMP or cyclic GMP had unmeasurable quantities of either compound. The data suggests that this unique action of cyclic CMP is not due to an effect of this nucleotide on adenyl cyclase or phosphodiesterase since intracellular levels of cyclic AMP were unchanged. There also appeared to be no unique binding proteins for this compound. It would appear that the cells are more permeable to cyclic CMP than to cyclic AMP or cyclic GMP and permit the accumulation of sufficient quantities of this nucleotide to reach intracellular concentrations capable of mimicking the effects of cyclic AMP. The data also suggest that the binding affinity and proteinkinase stimulating activity of a cyclic AMP analogue in partially purified systems may not be indicative of its “hormone-like” action on the intact cell. (Endocrinology93: 461, 1973)