Cortisol Secretion Pathway Figure 1. Time- and Concentration-dependent Effects of ACTH and 8CPT-29-OMe-cAMP on Cortisol Secretion. Bovine AZF cells were plated as described in the Methods. After 24 h, media was replaced with the same media without (control) or with 8CPT-29-OMe-cAMP or ACTH. A) Time- and concentration-dependent effects of 8CPT-29-OMe-cAMP on cortisol secretion. Media was sampled and cortisol measured at 4, 24 and 48 h after treating AZF cells with 8CPT-29-OMe-cAMP at concentrations ranging from 10 to 50 mM. B) Concentration-response curve for 8CPT-29-OMe- cAMP-stimulated on cortisol secretion after 24 h. Data were fit with an equation of the form: Y = min+(max2min)/(1+10‘((Log EC502X)*b)) where EC50 is the concentration that produces 50% of maximal effect and b is the Hill slope. C) Effect of 8CPT-29-OMe-cAMP (30 mM) and ACTH (2 nM) on cortisol secretion at 1, 2 or 3 h. D) Time course for ACTH (2 nM) and 8CPT-29-OMe-cAMP (30 mM). Media was sampled and cortisol determined at times from 8 to 96 h. doi:10.1371/journal.pone.0006088.g001 steroidogenic actions of 8CPT-29-OMe-cAMP were mediated by this compound rather than a metabolite, we measured the effect of the poorly hydrolyzable analog Sp-8CPT-29-OMe-cAMP on cortisol synthesis and the expression of steroid hydroxylase mRNAs. Although Sp-8CPT-29-OMe-cAMP activates Epac proteins with a potency similar to its hydrolyzable analog [30,35], the non-hydrolyzable compound at concentrations up to 100 mM was ineffective at increasing cortisol secretion or enhancing the expression of steroid hydroxylase mRNAs (Figure 3A,C,D). By comparison, at 100 mM, 8CPT-29-OMe- cAMP and its non-hydrolyzable analog activated Rap1 similarly (Figure 3B). These results indicated that the substantial increases in steroid hydroxylase mRNAs and cortisol secretion induced by 8CPT-29- OMe-cAMP were not mediated through Epac2. They further suggested that this response required the conversion of this ESCA to one or more active metabolites. Effect of Metabolites of 8CPT-29-OMe-cAMP on Steroidogenesis 8CPT-29-OMe-cAMP can be converted to 8CPT-29-OMe- 59AMP by cyclic nucleotide phosphodiesterase (see Figure 4) [30,36]. This metabolite stimulated cortisol secretion and expression of steroid hydroxylase with a temporal pattern, potency, and effectiveness similar to the parent ESCA (Figure 5). In the experiment illustrated, a 6 h exposure to 8CPT-29-OMe- 59AMP (100 mM) did not significantly increase cortisol synthesis. However, by 48 h, cortisol production increased 400 fold. Accordingly, after 48 h, 8CPT-29-OMe-59AMP (50 mM) had induced a nearly 50 fold (49.661.9, n = 3) increase in CYP17. PLoS ONE | www.plosone.org 4 June 2009 | Volume 4 | Issue 6 | e6088