human Ral-GDS-Rap binding domain bound to glutathione agarose (Ral GDS-RBD agarose) were performed using a Rap1 activation assay kit according to the manufacturer’s directions (Millipore, Billerica, MA). Briefly, ,106106 cells were plated on fibronectin-treated 10 cm plates in DMEM/F12+ for 48 h, changed to serum-free DMEM for 12 hours, directly stimulated with Epac activators for 15 min, washed twice with ice-cold TBS, then lysed in 600 ml of buffer containing 0.05 M Tris/HCl (pH 7.4), 0.5 M NaCl, 1% NP-40, 2.5 mM MgCl2, 5% glycerol, and protease inhibitors (Complete, EDTA-free, Roche Applied Science, Mannheim, Germany). Cells were disrupted by passing lysate five times through a 29-gauge needle; lysate was cleared by centrifugation at 14 000 g for 10 min at 4uC. An aliquot (60 ml) of the supernatant was reserved for estimation of total Rap1. Remaining supernatant was mixed with glutathione-agarose and incubated for 1 h at 4uC. Samples were then centrifuged at 10 000 g for 30 s at 4uC, washed 36 with lysis buffer, suspended in 40 ml of 26 Laemmle buffer containing 10 mM DTT, and separated by 8–16% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane, incubated with polyclonal anti-Rap1 antibodies (Millipore, Bill- erica, MA), and visualized by enhanced chemilluminescence (Pierce of Thermo Fisher Scientific, Rockford, IL). A-kinase Assay PKA activity was measured with a SignaTECT cAMP- dependent protein kinase assay kit (Promega, Madison, WI) in which PKA-dependent phosphorylation of biotinylated peptides can be quantitatively measured as a function of PKA activity. AZF cells were plated on 60-mm fibronectin-treated dishes in DMEM/ F12+ at a density of 46106 cells/dish. After 24 h, the media was replaced with either control media (DMEM/F12+) or the same media containing ACTH (1–24), 8CPT-29-OMe-cAMP, Sp- 8CPT-29-OMe-cAMP, or other agents as required. At the end of the incubation period, cells were washed 46 with ice-cold PBS, suspended in cold extraction buffer (25 mM Tris-HCl pH 7.4, 0.5 mM EGTA, 10 mM b-mercaptoethanol, 0.5 mM Pefabloc- SC (Roche Applied Science, Indianapolis, IN), and protease inhibitors with EDTA (Complete Mini protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN), 1 per 10 mls lysis solution). Lysates were homogenized using a cold Dounce homogenizer, then centrifuged for 5 min at 4uC, 14,0006 g. Each experimental condition was assayed in duplicate, and duplicate 5 ml samples of lysate supernatant from each were tested for PKA activity according to the manufacturer’s directions. Maximum activatable PKA activity was obtained for each respective sample by the addition of cAMP (5 mM) directly to the sample lysates before determining PKA activity according to the manufacturer’s directions. PKA values are presented as mean6SEM. Results 8CPT-29-OMe-cAMP induces a delayed increase in Cortisol Secretion by AZF Cells When applied to bovine AZF cells, the ESCA 8CPT-29-OMe- cAMP produced a remarkable concentration-dependent, delayed increase in cortisol secretion. As illustrated in Figure 1A, a 4 h exposure to 8CPT-29-OMe-cAMP at concentrations between 10 and 50 mM produced little or no increase in the quantity of cortisol secreted. However, by 24 h, large 4 to 15 fold increases in the quantity of cortisol produced were present. Between 24 and 48 h, the ESCA induced even greater increases in the rate of secretion; by 48 h, 8CPT-29-OMe-cAMP (50 mM) had increased the Cortisol Secretion Pathway quantity of cortisol produced more than 20 times the control value. 8CPT-29-OMe-cAMP stimulated cortisol synthesis with an EC50 of 23 mM (Figure 1B). ACTH stimulates cortisol secretion through rapid and delayed cAMP-dependent mechanisms that are incompletely understood [1,13]. Experiments with 8CPT-29-OMe-cAMP suggested that the delayed effects of ACTH on cortisol synthesis could be mediated through activation of Epac2. ACTH was compared to 8CPT-29- OMe-cAMP with respect to the time-dependent stimulation of cortisol production for periods from 1 to 96 h. While ACTH- induced increases in the rate of cortisol secretion were present by 1 h and subsided by 48 h, the delayed 8CPT-29-OMe-cAMP- stimulated increases in the rate of cortisol synthesis persisted for at least 96 h (Figure 1C,D). 8CPT-29-OMe-cAMP Stimulates Expression of Steroid Hydroxylase mRNAs The delay of many hours that preceded 8CPT-29-OMe-cAMP- stimulated cortisol secretion suggested that the response was mediated through an action on gene transcription. CYP11a1, CYP17, and CYP21 are three of the steroid hydroxylases that mediate the stepwise synthesis of cortisol from cholesterol. CYP11a1 is a mitochondrial enzyme that catalyses the synthesis of pregnenolone from cholesterol, the first and rate-limiting enzymatic step in cortisol synthesis. CYP17 and CYP21 are microsomal enzymes [6]. 8CPT-29-OMe-cAMP (10-50 mM) induced concentration-dependent increases in the expression of each of the three steroid hydroxylase mRNAs. In the experiment illustrated in Figure 2A, maximum increases ranged from over three fold (3.361.1, n = 7) for CYP11a1 to 20 fold (20.662.6, n = 6) for CYP17. The increases in CYP17 mRNA were typically greatest relative to time-matched controls since this is the only one of the steroid hydroxylases whose expression is reported to be entirely cAMP-dependent [33,34]. The 8CPT-29-OMe-cAMP- stimulated increases in the quantity of mRNAs for each of the steroid hydroxylases occurred only after a delay of more than 4 h, while mRNA levels continued to increase for periods up to 72 h (Figure 2B,C). The induction of the steroid hydroxylase mRNAs by the ESCA was not reduced by the PKA antagonist H-89. Overall, H-89 (10 mM) did not significantly affect 8CPT-29-OMe-cAMP-induced CYP17 and CYP11a1 expression (Figure 2B). The kinetics of ACTH and 8CPT-29-OMe-cAMP stimulation of steroid hydroxylase mRNA expression were consistent with their actions on cortisol secretion. In the experiments illustrated in Figure 2C, the kinetics of ACTH and 8CPT-29-OMe-cAMP induction of CYP17 mRNAs were compared at times from 0.5 to 30 h. ACTH induced large increases in CYP17 mRNA that were present after 4 h, and reached a maximum of 20 fold by 20 h. In contrast, 8CPT-29-OMe-cAMP stimulated a slower, delayed increase in CYP17 mRNA that was significant at 10 h, but had not reached a maximum by 30 h. Similar results were obtained for CYP11a, and CYP21 (data not shown). Sp-8CPT-29-OMe-cAMP does not Stimulate Cortisol Synthesis The ability of 8CPT-29-OMe-cAMP to induce large increases in the expression of steroid hydroxylase mRNAs and cortisol synthesis by bovine AZF cells suggested that the delayed actions of ACTH and cAMP on cortico-steroidogenesis are mediated through activation of Epac2. However, similar to cAMP, this ESCA is metabolized by cyclic nucleotide phosphodiesterase as well as other enzymes [30]. To determine whether the PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e6088