Cortisol Secretion Pathway Figure 8. Effects of 8CPT-29-OMe-cAMP and Metabolites are PKA-independent but Cycloheximide-sensitive. A) Effect on PKA activity. AZF cells were either untreated (control), or incubated with ACTH (2 nM), 8CPT-29-OMe-cAMP (30 mM), Sp-8CPT-29-OMe-cAMP (30 mM), 8CPT-29-OMe- ado (30 mM), or 8CPT-ade (30 mM) for 30 min after which lysates were collected and assayed for PKA activity as described in the Methods. PKA activity is expressed as mean6SEM of duplicate determinations from duplicate independent samples. B) Effect of cycloheximide on 8CPT-29-OMe-cAMP- dependent increases in steroidogenic and StAR mRNAs. AZF cells were either untreated (control, white bar), or incubated with cycloheximide (CH, 5 mg/ml, striped bar), 8CPT-29-OMe-cAMP (30 mM, grey bar), or 8CPT-29-OMe-cAMP plus CH (striped/grey bar), as indicated. Total RNA was isolated after 48 h. Membranes were hybridized with specific probes for StAR, CYP11a1, CYP17, or CYP21 mRNA. Each lane contained 10 mg of total RNA. StAR mRNA value was determined from % control for the 2.75 Kb band value. doi:10.1371/journal.pone.0006088.g008 Whatever the identity of the active metabolite, it appears certain that it acts through a signaling pathway that is different from those activated by cAMP. First, in contrast to cAMP which triggers both rapid and delayed increases in cortisol synthesis through PKA- dependent mechanisms, 8CPT-29-OMe-cAMP and its metabolites stimulate only delayed increases in cortisol without activating PKA [1,13,38]. The activation of Rap1 by 8CPT-29-OMe-cAMP and it non- hydrolyzable analog were not correlated with the expression of steroidogenic mRNAs or cortisol production. In fact, 8CPT-29- OMe-cAMP stimulated cortisol synthesis at concentrations that produced little or no Rap1 activation, while at higher concentra- tions, Sp-8CPT-29-OMe-cAMP activated Rap1 without increas- ing cortisol synthesis. These results argue against any role for Epac2 in cAMP-stimulated cortico-steroidogenesis. Overall, the findings of this study do not rule out the possibility that an ESCA metabolite functions through an unknown cAMP-independent pathway which may also be activated by ACTH. ACTH and an O-nitrophenyl sulphenyl derivative produce large increases in cortisol synthesis at concentrations that stimulate no measurable increase in cAMP production [39,40]. Alternatively, metabolite-sensing riboswitches, including those binding adenine and adenine derivatives, control gene expression in prokaryotes as well as eukaryotes [41,42]. It is not known whether hormone synthesis in eukaryotic cells could also be regulated by small molecules acting through riboswitches. ESCA metabolites could also activate unidentified orphan receptors. The delay of several hours and the cycloheximide-sensitivity indicate that 8CPT-29-OMe-cAMP-stimulated mRNA expression and cortisol secretion require de novo protein synthesis. It is possible that the active molecule induces the expression of a transcription factor that specifically enhances the transcription of genes coding for steroidogenic proteins. In this regard, we found that 8CPT-29-OMe-cAMP and its metabolites do not stimulate the PLoS ONE | www.plosone.org 10 June 2009 | Volume 4 | Issue 6 | e6088