Cortisol Secretion Pathway Figure 3. Sp-8CPT-29-OMe-cAMP activates RAP1A but does not increase either Cortisol Secretion or Steroidogenic Protein Gene Expression. AZF cells were either untreated (control), or incubated with 8CPT-29-OMe-cAMP, or Sp-8CPT-29-OMe-cAMP, as indicated. A) Comparison of effect of 8CPT-29-OMe-cAMP (50 mM) and Sp-8CPT-29-OMe-cAMP (30, 50, or 100 mM) on cortisol secretion after 48 h. B) Activation of Rap1 by 8CPT- 29-OMe-cAMP and Sp-8CPT-29-OMe-cAMP in AZF cells. For pull-down assays, AZF cells were prepared and cultured in 10 cm dishes as described in the Methods. Before lysis, cells were incubated for 15 min either without (control) or with 50 or 100 mM 8CPT-29-OMe-cAMP, or 100 mM SP-8CPT-29- OMe-cAMP. C) Sp-8CPT-29-OMe-cAMP does not increase CYP17 or CYP11a1 mRNA expression. AZF cells were incubated without (control, white bar) or with either 8CPT-29-OMe-cAMP (30 mM, grey bar), or Sp-8CPT-29-OMe-cAMP (30 mM, striped/grey bar). Total RNA was isolated after 48 h, electrophoresed, blotted and probed as described in the Methods. Each lane contained 10 mg of total RNA. Membranes were hybridized with specific probe for bovine CYP11a1 or CYP17, as indicated. D) Cortisol measurements from media samples from experiment illustrated in (C). doi:10.1371/journal.pone.0006088.g003 Figure 4. Structures of 8CPT-29-OMe-cAMP and Metabolites. Chemical structures of 8CPT-29-OMe-cAMP and its metabolites. doi:10.1371/journal.pone.0006088.g004 PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e6088