cAMP Analogs and Their Metabolites Enhance TREK-1 mRNA and K⁺Current Expression in Adrenocortical Cells

Abstract

bTREK-1 K ϩ channels set the resting membrane potential of bovine adrenal zona fasciculata (AZF) cells and function pivotally in the physiology of cortisol secretion. Adrenocorticotropic hormone controls the function and expression of bTREK-1 channels through signaling mechanisms that may involve cAMP and downstream effectors including protein kinase A (PKA) and exchange protein 2 directly activated by cAMP (Epac2). Using patch-clamp and Northern blot analysis, we explored the regulation of bTREK-1 mRNA and K ϩ current expression by cAMP analogs and several of their putative metabolites in bovine AZF cells. At concentrations sufficient to activate both PKA and Epac2, 8-bromoadenosine-cAMP enhanced the expression of both bTREK-1 mRNA and K ϩ current. N 6 -Benzoyladenosine-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K ϩ current measured at times from 24 to 96 h. An Epac-selective cAMP analog, 8-(4-chlorophenylthio)-2Ј-O-methyl-cAMP (8CPT-2Ј-OMe-cAMP), potently stimulated bTREK-1 mRNA and K ϩ current expression, whereas the nonhydrolyzable Epac activator 8-(4-chlorophenylthio)-2Ј-O-methyl-cAMP, Sp-isomer was ineffective. Metabolites of 8CPT-2Ј-OMe-cAMP, including 8-(4-chlorophenylthio)-2Ј-O-methyladenosine-5Ј-O-monophosphate and 8CPT-2Ј-OMe-adenosine, promoted the expression of bTREK-1 transcripts and ion current with a temporal pattern, potency, and effectiveness resembling that of the parent compound. Likewise, at low concentrations, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 30 M) but not its nonhydrolyzable analog 8-(4-chlorophenylthio)-cAMP, Sp-isomer, enhanced the expression of bTREK-1 mRNA and current. 8CPT-cAMP metabolites, including 8CPT-adenosine and 8CPT-adenine, also increased bTREK-1 expression. These results indicate that cAMP increases the expression of bTREK-1 mRNA and K ϩ current through a cAMP-dependent but Epac2-independent mechanism. They further demonstrate that one or more metabolites of 8-(4-chlorophenylthio)-cAMP analogs potently stimulate bTREK-1 expression by activation of a novel cAMP-independent mechanism. These findings raise significant questions regarding the specificity of 8-(4-chlorophenylthio)-cAMP analogs as cAMP mimetics.

ACTH Induces Cav3.2 Current and mRNA by cAMP-dependent and cAMP-independent Mechanisms

“…In patch clamp experiments, we found that, when included in the pipette solution at very low concentrations (i.e. Ͻ10 M), 6-Bnz-cAMP completely blocks the activity of bTREK-1 channels, even in the presence of several PKA antagonists (36).…”

“…5 show (38,39). At concentrations above 100 M, 6-Bnz-cAMP markedly enhances cortisol secretion (36). Treating bovine AZF cells with 6-Bnz-cAMP for prolonged periods failed to induce the expression of Ca v 3.2 current.…”

“…These results show that the mechanisms by which ACTH regulates the expression of Ca v 3.2 differ fundamentally from those that control the expression of steroid hydroxylases and bTREK-1 K ϩ channels. Specifically, ACTH-stimulated increases in cAMP alone appear to be sufficient to induce the expression of steroid hydroxylase and bTREK-1 mRNAs and their corresponding functional proteins (6,7,36).…”

“…Experiments were done to determine whether ACTH-stimulated expression of Ca v 3.2 current was mediated by cAMP. 8-Br-cAMP activates PKA and Epac proteins at concentrations above 100 M and stimulates rapid and delayed increases in cortisol secretion by bovine AZF cells (25,26,36). However, as illustrated in Fig.…”

“…8CPT-cAMP can be sequentially converted to 8CPT-5Ј-AMP, 8CPT-Ado, and 8CPT-Ade by the cellular enzymes cyclic nucleotide phosphodiesterase, 5Ј nucleotidases, and purine nucleotide phosphorylase, respectively (36,37).…”