cAMP analogs and their metabolites enhance TREK-1 mRNA and K+ current expression in adrenocortical cells

Abstract

bTREK-1 K(+) 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(+) 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(+) current. N(6)-Benzoyladenosine-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K(+) current measured at times from 24 to 96 h. An Epac-selective cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8CPT-2'-OMe-cAMP), potently stimulated bTREK-1 mRNA and K(+) current expression, whereas the nonhydrolyzable Epac activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, Sp-isomer was ineffective. Metabolites of 8CPT-2'-OMe-cAMP, including 8-(4-chlorophenylthio)-2'-O-methyladenosine-5'-O-monophosphate and 8CPT-2'-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 microM) 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(+) 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.

Fig. 1.

Long-term effect of adrenocorticotropin and 8-Br-cAMP on the expression of bTREK-1 current. AZF cells were used for patch-clamp experiments 1 to 48 h after plating. AZF cells were plated in media containing no further addition (control; A and B), 2 nM adrenocorticotropin (ACTH) (C), or 8-Br-cAMP (D). Whole-cell K⁺ currents were recorded in response to voltage steps to +20 mV applied from −80 mV at 30-s intervals with or without depolarizing prepulses to −20 mV. Pipettes contained standard solution (see Materials and Methods). A to D, representative K⁺ current traces recorded with (right traces) and without (left traces) depolarizing prepulses, and corresponding plot of bTREK-1 amplitudes with (○) and without (●) depolarizing pulses. Times indicated on traces correspond to those on the graph at right. E, summary of experiments as in A to D. Bars represent bTREK-1 current density expressed as mean ± S.E.M. of indicated number of determinations at 1 and 48 h in control media and after 48-h exposure to adrenocorticotropin (2 nM) or 8-Br-cAMP (300 μM), as indicated.

Fig. 2.

6-Bnz-cAMP induces the expression of bTREK-1 current. AZF cells were cultured in media containing no further addition (control) (A), 30 μM 6-Bnz-cAMP (B), or 200 μM 6-Bnz-cAMP (C). Whole-cell K⁺ currents were recorded from AZF cells in response to voltage steps to +20 mV applied from −80 mV at 30-s intervals with or without depolarizing prepulses to −20 mV. Pipettes contained standard solution (see Materials and Methods). A to C, representative K⁺ current traces recorded with (right traces) and without (left traces) depolarizing prepulses, and corresponding plot of bTREK-1 amplitudes with (○) and without (●) depolarizing pulses. Times indicated on traces correspond to those on the graph at right. Cells were either untreated (A) or were treated with 30 μM (B) or 200 μM (C) 6-Bnz-cAMP for 48 h before recording. D, summary of experiments as in A to C. Bars represent bTREK-1 current density expressed as mean ± S.E.M. of indicated number of determinations after 48 to 96 h of exposure to 6-Bnz-cAMP (30 or 300 μM), as indicated.

Fig. 3.

Effect of Epac2-selective cAMP analogs on bTREK-1 current expression. Whole-cell K⁺ currents were recorded from AZF cells in response to voltage steps to +20 mV applied from −80 mV at 30-s intervals with or without depolarizing prepulses to −20 mV. Pipettes contained standard solution (see Materials and Methods). A and B, representative K⁺ current traces recorded with (right traces) and without (left traces) depolarizing prepulses, and corresponding plot of bTREK-1 amplitudes with (○) and without (●) depolarizing pulses. Time indicated on traces corresponds to those plotted on graph at right. AZF cells with treated with 30 μM 8CPT-2′-OMe-cAMP (A) or 30 μM Sp-8CPT-2′-OMe-cAMP (B) for 48 h before recording. C, summary of experiments as in A and B: bars specify bTREK-1 current density expressed as mean ± S.E.M. of an indicated number of determinations after 24 to 96 h of exposure to 8CPT-2′-OMe-cAMP (30 μM) or Sp-8CPT-2′-OMe-cAMP (30 μM) as indicated.

Fig. 4.

Long-term effect of Sp-8CPT-2′-OMe-cAMP and 8CPT-2′-OMe-Ado on expression of bTREK-1 current. AZF cells were cultured for 48 h in media containing no further addition (control), Sp-8CPT-2′-OMe-cAMP (100 μM) (A), or 8CPT-2′-OMe-Ado (30 μM) (B). Whole-cell K⁺ currents were recorded from AZF cells in response to voltage steps to +20 mV applied from −80 mV at 30-s intervals with or without depolarizing prepulses to −20 mV. Pipettes contained standard solution (see Materials and Methods). A and B, representative K⁺ current traces recorded with (right traces) and without (left traces) depolarizing prepulses, and corresponding plot of bTREK-1 amplitudes with (○) and without (●) depolarizing pulses. Times indicated on traces correspond to those plotted on the graph at right. C, summary of experiments as in A and B: bars represent bTREK-1 current density expressed as mean ± S.E.M. of indicated number of determinations after 48 or 72 h of exposure to Sp-8CPT-2′-OMe-cAMP (100 μM) or 8CPT-2′-OMe-Ado (30 μM) as indicated.

Fig. 5.

Chemical structures of 8CPT- 2′-OMe-cAMP, 8CPT-cAMP, and their metabolites.

Fig. 6.

Effects of adrenocorticotropin, 8-Br-cAMP, and 6-Bnz-cAMP on bTREK-1 gene expression. AZF cells were incubated either without (control) or with adrenocorticotropin, 8-Br-cAMP, 6-Bnz-cAMP, or 6-Bnz-cAMP + H-89, as indicated. Total RNA was isolated as described under Materials and Methods. Membranes were hybridized with specific probe for bTREK-1. 18S rRNA bands from representative gels are shown as evidence of even loading. bTREK-1 mRNA levels are expressed as percent of the 3.6-kb control band value. A, effect of time in culture on bTREK-1 mRNA. AZF cells were plated and total RNA isolated after 5, 24, or 48 h in culture, as indicated. B, effect of adrenocorticotropin (ACTH) and 8-Br-cAMP on bTREK-1 mRNA expression. AZF cells were cultured overnight before either no addition (control, □) or addition of adrenocorticotropin (2 nM, ■) or 8-Br-cAMP (30 μM, light gray bar; 300 μM, dark gray bar) for 48 h before isolating total RNA. *, statistically significant difference between control and treated cells (*, P < 0.02). C, effect of 6-Bnz-cAMP and H-89 on bTREK-1 mRNA. AZF cells were plated and cultured overnight before either no addition (control, open bar) or addition of H-89 (10 μM, striped bar), 6-Bnz-cAMP (200 μM, gray bar), or 6-Bnz-cAMP + H-89 (gray striped bar) for 24 h before isolating total RNA. Cells were preincubated with H-89 (10 μM) for 1 h before the 6-Bnz-cAMP (200 μM) addition (*, P < 0.03).

Fig. 7.

Effects of 8CPT-2′-OMe-cAMP and its metabolites on bTREK-1 gene expression. AZF cells were cultured overnight and then incubated either without (control) or with 8CPT-2′-OMe-cAMP, Sp-8CPT-2′-OMe-cAMP, 8CPT-2′-OMe-5′AMP, or 8CPT-2′-OMe-Ado as indicated. Total RNA was isolated as described under Materials and Methods. Membranes were hybridized with a specific probe for bTREK-1; bTREK-1 mRNA levels are expressed as a percentage of the 3.6-kb control band value. 18S rRNA bands from representative gels are shown as evidence of even loading. A, concentration-dependent effect of 8CPT-2′-OMe-cAMP on bTREK-1 mRNA expression. AZF cells were untreated (control, □) or treated with 8CPT-2′-OMe-cAMP (1–50 μM, ) for 48 h before isolating total RNA. B, time-dependent effect of 8CPT-2′-OMe-cAMP on bTREK-1 mRNA expression. AZF cells were either untreated (control, □) or treated with 30 μM 8CPT-2′-OMe-cAMP () for 1 to 48 h before isolating total RNA. C, effect of 8CPT-2′-OMe-cAMP, Sp-8CPT-2′-OMe-cAMP, and 8-Br-cAMP on bTREK-1 mRNA expression. AZF cells were either untreated (control, open bar) or treated with 30 μM 8CPT-2′-OMe-cAMP (gray bar, left), 30 μM Sp-8CPT-2′-OMe-cAMP (gray striped bar, left), 100 μM Sp-8CPT-2′-OMe-cAMP (gray striped bar, right), or 300 μM 8-Br-cAMP (black bar, right) for 48 h before isolating total RNA (*, P < 0.005). D, effect of metabolites of 8CPT-2′-OMe-cAMP on induction of bTREK-1 mRNA. AZF cells were treated with either 8CPT-2′-OMe-5′AMP (0.1–100 μM, gray dotted bars), 8CPT-2′-OMe-Ado (0.1–50 μM, dark gray bars). or adenosine (50 μM, light gray bar) for 48 h before isolating total RNA.

Fig. 8.

Long-term effect of 8CPT-cAMP and Sp-8CPT-cAMP on the expression of bTREK-1 current. AZF cells were cultured overnight and then incubated either without (control) or with 8CPT-cAMP (30 μM) or Sp-8CPT-cAMP (30 or 300 μM), as indicated. Whole-cell K⁺ currents were recorded from AZF cells in response to voltage steps to +20 mV applied from −80 mV at 30-s intervals with or without depolarizing prepulses to −20 mV. Pipettes contained standard solution as described under Materials and Methods. A to C, representative K⁺ current traces recorded with (right traces) and without (left traces) depolarizing prepulses and corresponding plot of bTREK-1 amplitudes with (○) and without (●) depolarizing pulses. Time indicated on traces corresponds to those plotted on the graph at right. Cells were treated with 30 μM 8CPT-cAMP (A), 30 μM Sp-8CPT-2′-OMe-cAMP (B), or 300 μM Sp-8CPT-cAMP (C) for 48 h before recording. D, summary of experiments as in A to C. Bars represent bTREK-1 current density expressed as mean ± S.E.M. of indicated number of determinations after 48-h exposure to 8CPT-cAMP (30 μM) or Sp-8CPT-2′-OMe-cAMP (30 or 300 μM) as indicated.

Fig. 9.

Metabolites of 8CPT-cAMP induce bTREK-1 current. AZF cells were cultured overnight then incubated either without (control) or with 8CPT-Ado (30 μM) or 8CPT-Ade (30 μM) as indicated. Whole-cell K⁺ currents were recorded from AZF cells in response to voltage steps to +20 mV applied from −80 mV at 30-s intervals with or without depolarizing prepulses to −20 mV. Pipettes contained standard solution as described under Materials and Methods. A and B, representative K⁺ current traces recorded with (right traces) and without (left traces) depolarizing prepulses and corresponding plot of bTREK-1 amplitudes with (○) and without (●) depolarizing pulses. Time indicated on traces corresponds to those plotted on graph at right. Cells were treated with 30 μM 8CPT-Ado (A) or 30 μM 8CPT-Ade (B) for 48 h before recording. C, summary of experiments as in A and B. Bars represent bTREK-1 current density expressed as mean ± S.E.M. of indicated number of determinations after 48-h exposure to 8CPT-Ado or 8CPT-Ade as indicated.

Fig. 10.

Effects of 8CPT-cAMP, Sp-cAMP, and 8CPT-Ade on expression of bTREK-1 mRNA. AZF cells were cultured overnight then incubated either without (control) or with 8CPT-cAMP, Sp-cAMP, 8CPT-Ade, or adrenocorticotropin as indicated. Total RNA was isolated as described under Materials and Methods. Membranes were hybridized with a specific probe for bTREK-1; bTREK-1 mRNA levels are expressed as a percentage of the 3.6-kb control band value.18S rRNA bands from representative gels are shown as evidence of even loading. A, 8CPT-cAMP but not Sp-8CPT-cAMP induces bTREK-1 mRNA. AZF cells were either untreated (control, open bar) or treated with 30 μM 8CPT-cAMP (dark gray bar) or 30 μM Sp-8CPT-cAMP (dark gray striped bar) for 48 h before isolating total RNA (*, P < 0.02). B, concentration-dependent effect of 8CPT-Ade on bTREK-1 mRNA. AZF cells were either untreated (control, □) or treated with 8CPT-Ade (1–50 μM, ) for 48 h before isolating total RNA (*, P < 0.001). C, time-dependent effect of 8CPT-Ade and adrenocorticotropin (ACTH) on bTREK-1 mRNA expression. AZF cells were either untreated (control, □) or treated with 30 μM 8CPT-cAMP () or 2 nM adrenocorticotropin (■) for 5, 24, and 48 h before isolating total RNA (*, P < 0.001).