The steroidogenic actions of cAMP, including the rapid and delayed stimulation of cortisol synthesis, have long been attributed to the activation of PKA; however, confounding evidence exists. Specifically, genes that code for steroidogenic enzymes in AZF cells are induced by cAMP, yet nearly all of these, regardless of species, lack consensus cAMP response elements in the 59 flanking region and therefore do not bind PKA phosphorylated transcrip- tion factors [8,20–24]. Further, PKA-stimulated transcription typically occurs with rapid kinetics and does not require de novo protein synthesis [25]. In contrast, ACTH- or cAMP-induced increases in steroid hydrox- ylase-specific mRNAs are cycloheximide sensitive and require periods of up to several hours before they are observed [8,20,25]. Therefore, ACTH and cAMP may increase the transcription of steroidogenic enzymes, and stimulate cortisol synthesis by novel PKA-independent mechanisms. In this regard, PKA-independent actions of ACTH and cAMP have been reported in AZF cells. cAMP inhibits bTREK-1 K+ channels in AZF cells by a mechanism that is independent of PKA, but mimicked by Epac-specific cAMP analogs [19,26]. ACTH enhances the expression of T-type Ca2+ channels in rat AZF cells by an H-89 insensitive mechanism [27]. Elucidating the signaling pathways by which ACTH and cAMP regulate corticosteroid synthesis has been hampered by the lack of specific reagents that could independently modulate PKA and Epacs. Exploiting differences in the cAMP-binding domains of these proteins, rational drug design has recently been used to synthesize cAMP derivatives that, at appropriate concentrations, selectively activate Epacs or PKA. These derivatives provide the opportunity to independently study cAMP-activated proteins in the regulation of cell function [28–30]. One of these, 8CPT-29-OMe-cAMP, selectively activates Epacs over PKA, and has been used extensively to determine the role of Epac1 and Epac2 in cell function [28,30]. However, in the present study we found that, in AZF cells, 8CPT-29-OMe-cAMP stimulated large, delayed increases in the expression of steroid hydroxylase mRNAs and cortisol synthesis by an Epac-indepen- dent mechanism. These effects were mediated by one or more metabolites of the parent compound through an unknown signaling pathway. Methods Materials DMEM/F12, antibiotics, fibronectin, and fetal bovine sera (FBS) were obtained from Invitrogen (Carlsbad, CA). Ultrahyb was purchased from Ambion (Austin, TX) and Prime-It II kit for random priming was purchased from Stratagene (La Jolla, CA). Phosphate buffered saline (PBS), tocopherol, selenite, ascorbic acid, and ACTH (1–24) were obtained from Sigma (St. Louis. MO). 8-(4-chlorophenylthio)-29-O-methyl-cAMP (8CPT-29-OMe- cAMP -Biolog #C041), hydrolysis-resistant 8-(4-chlorophe- nylthio)-29-O-methyl-cAMPs, Sp-isomer (Sp-8CPT-29-OMe- cAMP - Biolog # C052), 8-(4-chlorophenylthio)-29-O-methylade- nosine-59-O-monophosphate (8CPT-2-OMe-59AMP – Biolog # C078), 8-(4-chlorophenylthio)-29-O-methyladenosine (8CPT- 29OMe-Ado – Biolog #C070), and 8-(4-chlorophenylthio) ade- nine (8CPT-Ade – Biolog #C069) were purchased from Axxora,LLC (San Diego, CA). Cortisol EIA kit was from Diagnostic Systems Laboratories (Webster, TX). Rap1 activation assay kit was purchased from Millipore (Billerica, MA) and SignaTECT cAMP-dependent protein kinase assay kit was from Promega (Madison, WI). Cortisol Secretion Pathway Isolation and Culture of AZF Cells Bovine adrenal glands were obtained from steers (age 2–3 yr) at a local slaughterhouse. AZF cells were isolated as previously described [31]. After isolation, cells were either resuspended in DMEM/F12 (1:1) with 10% FBS, 100 U/ml penicillin, 0.1 mg/ ml streptomycin, and the antioxidants 1 mM tocopherol, 20 nM selenite and 100 mM ascorbic acid (DMEM/F12+) and plated for immediate use, or resuspended in FBS/5% DMSO, divided into aliquots, and stored in liquid nitrogen for future use. To ensure attachment when plating cells, dishes were treated with fibronectin (10 mg/ml) for 30 minutes then rinsed with warm, sterile PBS before adding cells. Cells were maintained at 37uC in a humidified atmosphere of 95% air-5% CO2. For experiments where cortisol alone was measured, cells were plated at a density of 0.5–1.06106 cells/35 mm dish. For experiments where cortisol was measured and RNA was isolated from cell lysates, cells were plated at a density of 5–76106 cells/60 mm culture dish. For all experiments except those for Rap1, cells were plated 24 h before incubating with ESCAs or metabolites. For Rap1 assay, cells were plated 48 h before incubating with 8CPT-29-OMe-cAMP or Sp-8CPT-29- OMe-cAMP. Cortisol Assay Media from experiments was either assayed immediately after collection or frozen (220uC) until all samples were available. Each experimental condition was assayed in duplicate and duplicate media samples from each were measured using a Cortisol EIA (DSL-10-200) from Diagnostic Systems Laboratories (Webster, TX), according to the manufacturer’s directions. If necessary, media samples were diluted using DMEM/F12+. Cortisol values are expressed as mean6SEM of duplicate independent determi- nations, assayed in duplicate. Northern Blot and Measurement of mRNA Total RNA isolation and Northern blot procedures have been described previously [32]. Briefly, 5–76106 AZF cells were plated on 60-mm fibronectin-treated dishes in DMEM/F12+. After 24 h, the serum-supplemented media were removed and 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, total RNA was extracted using RNeasy columns (Qiagen, Valencia, CA), electrophoresed on a denaturing gel, and transferred to a nylon transfer membrane (GeneScreen Plus, PerkinElmer Life Sciences, Waltham, MA). Probes were generated by RT-PCR using AMV reverse transcriptase (Pro- mega, Madison WI), specific primers, and total RNA isolated from bovine AZF cells as described above. Specific probes generated were as follows: StAR- bases 104-1047 of NM_174189, CYP11a1- bases 679-1816 of NM_176644, CYP17 - bases 68-755 of NM_174303, CYP21 – bases 491-1240 of NM_001013596. Probes were labeled with [a-32P]dCTP by random primer labeling (Prime-It II, Stratagene, La Jolla, CA). Northern autoradiograms were imaged using a Typhoon 9200 variable mode imager and quantitated using ImageQuant TL v2003.3 software (GE Healthcare Life Sciences, Piscataway, NJ). mRNA values are presented as mean6SEM of at least 3 independent determina- tions. For the figures, a representative Northern blot is shown for each set of experiments. Rap1 Activation Assay Rap1 activation assays using a glutathione reductase-tagged fusion protein corresponding to amino acids 788–884 of the PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e6088