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ACTH inhibits bTREK-1 K+ channels through multiple cAMP-dependent signaling pathways.

Liu H, Enyeart JA, Enyeart JJ - J. Gen. Physiol. (2008)

Bottom Line: The selective Epac activator, 8-pCPT-2'-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC(50) = 0.63 microM) at concentrations that did not activate PKA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA.

ABSTRACT
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 Ca(2+) 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 Ca(2+)-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 (IC(50) = 0.63 microM) 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.

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Inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP in twice-patched cells. Whole cell K+ currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from −80 mV, with or without depolarizing prepulses. Cells were sequentially patched with two pipettes containing standard solution, or the same solution supplemented with PKI(6–22) amide (4 μM), H-89 (10 μM), or 8-pCPT-2′-O-Me-cAMP (EA) as indicated. When bTREK-1 reached a stable maximum, the first pipette was withdrawn and the cell patched again with the second pipette. (A–C) Current traces and corresponding plots of bTREK-1 amplitude against time for cells patch clamped with pipettes containing the additions indicated. Closed circles represent pipette #1, closed triangles, pipette #2. Numbers on traces at left correspond to those on plot at right. Break in graph denotes time required to change patch pipettes.
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fig8: Inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP in twice-patched cells. Whole cell K+ currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from −80 mV, with or without depolarizing prepulses. Cells were sequentially patched with two pipettes containing standard solution, or the same solution supplemented with PKI(6–22) amide (4 μM), H-89 (10 μM), or 8-pCPT-2′-O-Me-cAMP (EA) as indicated. When bTREK-1 reached a stable maximum, the first pipette was withdrawn and the cell patched again with the second pipette. (A–C) Current traces and corresponding plots of bTREK-1 amplitude against time for cells patch clamped with pipettes containing the additions indicated. Closed circles represent pipette #1, closed triangles, pipette #2. Numbers on traces at left correspond to those on plot at right. Break in graph denotes time required to change patch pipettes.

Mentions: Control experiments showed that AZF cells could often be consecutively patched with two pipettes without compromising the recording of bTREK-1 currents. In the experiment illustrated in Fig. 8 A, the cell was consecutively patched by two pipettes containing standard pipette solution. bTREK-1 current amplitude remained relatively constant upon voltage clamping the cell with the second pipette. In contrast, when the second pipette contained 8-pCPT-2′-O-Me-cAMP (15 μM), bTREK-1 was rapidly inhibited (Fig. 8 B). Similar results were obtained in each of four experiments.


ACTH inhibits bTREK-1 K+ channels through multiple cAMP-dependent signaling pathways.

Liu H, Enyeart JA, Enyeart JJ - J. Gen. Physiol. (2008)

Inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP in twice-patched cells. Whole cell K+ currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from −80 mV, with or without depolarizing prepulses. Cells were sequentially patched with two pipettes containing standard solution, or the same solution supplemented with PKI(6–22) amide (4 μM), H-89 (10 μM), or 8-pCPT-2′-O-Me-cAMP (EA) as indicated. When bTREK-1 reached a stable maximum, the first pipette was withdrawn and the cell patched again with the second pipette. (A–C) Current traces and corresponding plots of bTREK-1 amplitude against time for cells patch clamped with pipettes containing the additions indicated. Closed circles represent pipette #1, closed triangles, pipette #2. Numbers on traces at left correspond to those on plot at right. Break in graph denotes time required to change patch pipettes.
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483331&req=5

fig8: Inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP in twice-patched cells. Whole cell K+ currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from −80 mV, with or without depolarizing prepulses. Cells were sequentially patched with two pipettes containing standard solution, or the same solution supplemented with PKI(6–22) amide (4 μM), H-89 (10 μM), or 8-pCPT-2′-O-Me-cAMP (EA) as indicated. When bTREK-1 reached a stable maximum, the first pipette was withdrawn and the cell patched again with the second pipette. (A–C) Current traces and corresponding plots of bTREK-1 amplitude against time for cells patch clamped with pipettes containing the additions indicated. Closed circles represent pipette #1, closed triangles, pipette #2. Numbers on traces at left correspond to those on plot at right. Break in graph denotes time required to change patch pipettes.
Mentions: Control experiments showed that AZF cells could often be consecutively patched with two pipettes without compromising the recording of bTREK-1 currents. In the experiment illustrated in Fig. 8 A, the cell was consecutively patched by two pipettes containing standard pipette solution. bTREK-1 current amplitude remained relatively constant upon voltage clamping the cell with the second pipette. In contrast, when the second pipette contained 8-pCPT-2′-O-Me-cAMP (15 μM), bTREK-1 was rapidly inhibited (Fig. 8 B). Similar results were obtained in each of four experiments.

Bottom Line: The selective Epac activator, 8-pCPT-2'-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC(50) = 0.63 microM) at concentrations that did not activate PKA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA.

ABSTRACT
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 Ca(2+) 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 Ca(2+)-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 (IC(50) = 0.63 microM) 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.

Show MeSH
Related in: MedlinePlus