Cortisol Secretion Pathway Figure 6. 8CPT-29-OMe-Adenosine and 8CPT-Adenine Increase Cortisol Secretion and CYP17 mRNA Expression. AZF cells were incubated either without (control), or with 8CPT-29-OMe-Ado, adenosine, or 8CPT-Ade at concentrations from 1-50 mM. Media was sampled for cortisol measurements at several time points and total RNA was isolated after 48 h for measurement of CYP17 mRNA. A,B) Time- and concentration- dependent effects of 8CPT-29-OMe-Ado on cortisol secretion and CYP17 mRNA. A) Media was sampled and cortisol measured at 6, 24 and 48 h after incubating AZF cells without (control, white bars) or with 8CPT-29-OMe-Ado at concentrations ranging from 1 to 50 mM (grey bars) or adenosine (50 mM, black bar). B) After 48 h, mRNA was isolated and CYP17 mRNA measured by Northern blot. C, D) Time- and concentration-dependent effects of 8CPT-Ade on cortisol secretion and CYP17 mRNA. C) Media was sampled and cortisol determined at 6, 24 and 48 h after treating AZF cells without (control, white bars) or with 8CPT-Ade at concentrations ranging from 1 to 50 mM (dark grey bars). D) After 48 h, mRNA was isolated and CYP17 mRNA was measured by Northern blot. doi:10.1371/journal.pone.0006088.g006 signaling pathway. The function of Epac2 in bovine AZF cell physiology remains unknown. 8CPT-29-OMe-cAMP and Metabolites Initial experiments in which the ESCA induced pronounced, delayed increases in the expression of steroid hydroxylase and StAR mRNAs and associated cortisol synthesis led us to believe that these effects were mediated through activation of Epac2 which is robustly expressed in bovine AZF cells [19]. The failure of Sp-8CPT-29-OMe-cAMP to mimic the actions of its hydrolyzable analog was unexpected and appears to eliminate the possibility that ACTH- or cAMP-stimulated increases in cortisol synthesis are mediated in part by Epac2. These results also suggested that 8CPT-29-OMe-cAMP functions in this instance only after conversion to one or more bioactive metabolites. Accordingly, we found that three potential metabolites of the ESCA mimicked its effects on cortisol secretion and gene expression. Enzymes that could catalyze the conversion of 8CPT-29-OMe-cAMP to each of these presump- tive metabolites are expressed by mammalian cells. These include cyclic nucleotide phosphodiesterases, 59nucleotidases, hypoxanthine phosphoribosyltransferase, and nucleotide phos- phorylases [36]. Receptors and Signaling Pathways Currently, the active metabolites, associated receptor, and signaling pathway(s) that mediate the increases in cortisol synthesis and gene expression observed in this study have not been identified. It will be important to learn if 8CPT-Ade is further metabolized to an active compound. PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e6088