Metabolites of an Epac-Selective cAMP Analog Induce Cortisol Synthesis by Adrenocortical Cells through a cAMP-Independent Pathway

Adrenal zona fasciculata (AZF) cells express a cAMP-activated guanine nucleotide exchange protein (Epac2) that may function in ACTH-stimulated cortisol synthesis. Experiments were done to determine whether cAMP analogs that selectively activate Epacs could induce cortisol synthesis and the expression of genes coding for steroidogenic proteins in bovine AZF cells. Treatment of AZF cells with the Epac-selective cAMP analog (ESCA) 8CPT-2′-OMe-cAMP induced large (>100 fold), concentration-dependent, delayed increases in cortisol synthesis and the expression of mRNAs coding for the steroid hydroxylases CYP11a1, CYP17, CYP21, and the steroid acute regulatory protein (StAR). However, a non-hydrolyzable analog of this ESCA, Sp-8CPT-2′-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-2′-OMe-cAMP, including 8CPT-2′-OMe-5′AMP, 8CPT-2′-OMe-adenosine, and 8CPT-adenine all induced cortisol synthesis and steroid hydroxylase mRNA expression with a temporal pattern, potency, and effectiveness similar to the parent compound. At concentrations that markedly stimulated cortisol production, none of these metabolites significantly activated cAMP-dependent protein kinase (PKA). These results show that one or more metabolites of the ESCA 8CPT-2′-OMe-cAMP induce cortico-steroidogenesis by activating a panel of genes that code for steroidogenic proteins. The remarkable increases in cortisol synthesis observed in this study appear to be mediated by a novel cAMP-, Epac- and PKA-independent signaling pathway

ACTH Inhibits bTREK-1 K+ Channels through Multiple cAMP-dependent Signaling Pathways

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K+ channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca2+ entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI(6–22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca2+-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2′-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC50 = 0.63 μM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3–4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2′-O-Me-cAMP also inhibit these K+ channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH

Effects of 8CPT-2′-OMe-cAMP, Metabolites, and Sp-8CPT-2′-OMe-cAMP on StAR mRNA Expression.

<p>AZF cells were incubated either without (control), or with 8CPT-2′-OMe-cAMP, 8CPT-2′-OMe-5′AMP, Sp-8CPT-2′-OMe-cAMP, or 8CPT-Ade at indicated times. Total RNA was isolated after indicated times. Membranes were hybridized with specific probe for bovine StAR mRNA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006088#s2" target="_blank">Methods</a>. Each lane contained 10 µg of total RNA. 18S rRNA bands from representative gels are shown as evidence of even loading. A) Concentration-dependent effect of 8CPT-2′-OMe-cAMP on StAR mRNA. AZF cells were incubated without (control, white bar) or with 10–50 µM 8CPT-2′-OMe-cAMP (grey bars) for 48 hr before isolating total RNA. B) Time-dependent effects of 8CPT-2′-OMe-cAMP on StAR mRNA. AZF cells were incubated without (control, white bar), or with 8CPT-2′-OMe-cAMP (50 µM, grey bars) for indicated times after which total RNA was isolated. mRNA is expressed as % control value at 72 h. C) Comparison of effects of 8CPT-2′-OMe-cAMP, 8CPT-2′-OMe-5′AMP, and hydrolysis-resistant Sp-8CPT-2′-OMe-cAMP on StAR mRNA. AZF cells were incubated without (control, white bar) or with either 8CPT-2′-OMe-cAMP (30 µM, light grey bar), 8CPT-2′-OMe-5′AMP (30 µM, dark grey bar), or Sp-8CPT-2′-OMe-cAMP (30 µM, striped/grey bar) for 48 h after which total RNA was isolated. D) Concentration-dependent effect of 8CPT-Ade on StAR mRNA. AZF cells were incubated without (control, white bar) or with 10–50 µM 8CPT-Ade (grey bars) for 48 hr before isolating total RNA.</p

Sp-8CPT-2′-OMe-cAMP activates RAP1A but does not increase either Cortisol Secretion or Steroidogenic Protein Gene Expression.

<p>AZF cells were either untreated (control), or incubated with 8CPT-2′-OMe-cAMP, or Sp-8CPT-2′-OMe-cAMP, as indicated. A) Comparison of effect of 8CPT-2′-OMe-cAMP (50 µM) and Sp-8CPT-2′-OMe-cAMP (30, 50, or 100 µM) on cortisol secretion after 48 h. B) Activation of Rap1 by 8CPT-2′-OMe-cAMP and Sp-8CPT-2′-OMe-cAMP in AZF cells. For pull-down assays, AZF cells were prepared and cultured in 10 cm dishes as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006088#s2" target="_blank">Methods</a>. Before lysis, cells were incubated for 15 min either without (control) or with 50 or 100 µM 8CPT-2′-OMe-cAMP, or 100 µM SP-8CPT-2′-OMe-cAMP. C) Sp-8CPT-2′-OMe-cAMP does not increase CYP17 or CYP11a1 mRNA expression. AZF cells were incubated without (control, white bar) or with either 8CPT-2′-OMe-cAMP (30 µM, grey bar), or Sp-8CPT-2′-OMe-cAMP (30 µM, striped/grey bar). Total RNA was isolated after 48 h, electrophoresed, blotted and probed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006088#s2" target="_blank">Methods</a>. Each lane contained 10 µg 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).</p

Structures of 8CPT-2′-OMe-cAMP and Metabolites.

<p>Chemical structures of 8CPT-2′-OMe-cAMP and its metabolites.</p

8CPT-2′-OMe-Adenosine and 8CPT-Adenine Increase Cortisol Secretion and CYP17 mRNA Expression.

<p>AZF cells were incubated either without (control), or with 8CPT-2′-OMe-Ado, adenosine, or 8CPT-Ade at concentrations from 1-50 µM. Media was sampled for cortisol measurements at several time points and total RNA was isolated after 48 h for measurement of CYP17 mRNA. A,B) Time- and concentration-dependent effects of 8CPT-2′-OMe-Ado on cortisol secretion and CYP17 mRNA. A) Media was sampled and cortisol measured at 6, 24 and 48 h after incubating AZF cells without (control, white bars) or with 8CPT-2′-OMe-Ado at concentrations ranging from 1 to 50 µM (grey bars) or adenosine (50 µM, black bar). B) After 48 h, mRNA was isolated and CYP17 mRNA measured by Northern blot. C, D) Time- and concentration-dependent effects of 8CPT-Ade on cortisol secretion and CYP17 mRNA. C) Media was sampled and cortisol determined at 6, 24 and 48 h after treating AZF cells without (control, white bars) or with 8CPT-Ade at concentrations ranging from 1 to 50 µM (dark grey bars). D) After 48 h, mRNA was isolated and CYP17 mRNA was measured by Northern blot.</p

Time- and Concentration-dependent Effects of ACTH and 8CPT-2′-OMe-cAMP on Cortisol Secretion.

<p>Bovine AZF cells were plated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006088#s2" target="_blank">Methods</a>. After 24 h, media was replaced with the same media without (control) or with 8CPT-2′-OMe-cAMP or ACTH. A) Time- and concentration-dependent effects of 8CPT-2′-OMe-cAMP on cortisol secretion. Media was sampled and cortisol measured at 4, 24 and 48 h after treating AZF cells with 8CPT-2′-OMe-cAMP at concentrations ranging from 10 to 50 µM. B) Concentration-response curve for 8CPT-2′-OMe-cAMP-stimulated on cortisol secretion after 24 h. Data were fit with an equation of the form: Y = min+(max−min)/(1+10^∧((Log EC₅₀−X)*b)) where EC₅₀ is the concentration that produces 50% of maximal effect and <i>b</i> is the Hill slope. C) Effect of 8CPT-2′-OMe-cAMP (30 µM) and ACTH (2 nM) on cortisol secretion at 1, 2 or 3 h. D) Time course for ACTH (2 nM) and 8CPT-2′-OMe-cAMP (30 µM). Media was sampled and cortisol determined at times from 8 to 96 h.</p