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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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Effect of 8-pCPT-cAMP on bTREK-1 currents in perforated patch recordings. (A and B) Perforated patch recordings: whole cell K+ currents were recorded in the nystatin perforated patch configuration in response to voltage steps applied at 30-s intervals from a holding potential of −80 to +20 mV with (right traces) or without (left traces) depolarizing prepulses. After bTREK-1 reached a stable amplitude, cells were superfused with 8-pCPT-2′-O-Me-cAMP (designated EA for Epac activator) (30 μM), ACTH (200 pM), or 8-pCPT-cAMP (30 or 300 μM), as indicated. Numbers on current traces correspond to those on plot at right. (C) Summary of experiments as in A and B. Bars indicate mean ± SEM of indicated number of determinations.
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fig5: Effect of 8-pCPT-cAMP on bTREK-1 currents in perforated patch recordings. (A and B) Perforated patch recordings: whole cell K+ currents were recorded in the nystatin perforated patch configuration in response to voltage steps applied at 30-s intervals from a holding potential of −80 to +20 mV with (right traces) or without (left traces) depolarizing prepulses. After bTREK-1 reached a stable amplitude, cells were superfused with 8-pCPT-2′-O-Me-cAMP (designated EA for Epac activator) (30 μM), ACTH (200 pM), or 8-pCPT-cAMP (30 or 300 μM), as indicated. Numbers on current traces correspond to those on plot at right. (C) Summary of experiments as in A and B. Bars indicate mean ± SEM of indicated number of determinations.

Mentions: These PKA activity measurements demonstrated that 8-pCPT-2′-O-Me-cAMP could be used in bovine AZF cells as a selective Epac activator at concentrations up to 30 μM. To determine whether 8-pCPT-2′-O-Me-cAMP could inhibit bTREK-1 independently of PKA, AZF cells were superfused with this agent while recording bTREK-1 currents with the nystatin perforated patch technique (Horn and Marty, 1988). By using this technique, we hoped to minimize cell dialysis allowing 8-pCPT-2′-O-Me-cAMP to reach a higher intracellular concentration. However, in these experiments, 8-pCPT-2′-O-Me-cAMP (30 μM) inhibited bTREK-1 by only 10.2 ± 6.4% (n = 5), while ACTH (200 pM) inhibited bTREK-1 under the same conditions by 83.8 ± 2.8% (n = 7) (Fig. 5, A and C).


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

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

Effect of 8-pCPT-cAMP on bTREK-1 currents in perforated patch recordings. (A and B) Perforated patch recordings: whole cell K+ currents were recorded in the nystatin perforated patch configuration in response to voltage steps applied at 30-s intervals from a holding potential of −80 to +20 mV with (right traces) or without (left traces) depolarizing prepulses. After bTREK-1 reached a stable amplitude, cells were superfused with 8-pCPT-2′-O-Me-cAMP (designated EA for Epac activator) (30 μM), ACTH (200 pM), or 8-pCPT-cAMP (30 or 300 μM), as indicated. Numbers on current traces correspond to those on plot at right. (C) Summary of experiments as in A and B. Bars indicate mean ± SEM of indicated number of determinations.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effect of 8-pCPT-cAMP on bTREK-1 currents in perforated patch recordings. (A and B) Perforated patch recordings: whole cell K+ currents were recorded in the nystatin perforated patch configuration in response to voltage steps applied at 30-s intervals from a holding potential of −80 to +20 mV with (right traces) or without (left traces) depolarizing prepulses. After bTREK-1 reached a stable amplitude, cells were superfused with 8-pCPT-2′-O-Me-cAMP (designated EA for Epac activator) (30 μM), ACTH (200 pM), or 8-pCPT-cAMP (30 or 300 μM), as indicated. Numbers on current traces correspond to those on plot at right. (C) Summary of experiments as in A and B. Bars indicate mean ± SEM of indicated number of determinations.
Mentions: These PKA activity measurements demonstrated that 8-pCPT-2′-O-Me-cAMP could be used in bovine AZF cells as a selective Epac activator at concentrations up to 30 μM. To determine whether 8-pCPT-2′-O-Me-cAMP could inhibit bTREK-1 independently of PKA, AZF cells were superfused with this agent while recording bTREK-1 currents with the nystatin perforated patch technique (Horn and Marty, 1988). By using this technique, we hoped to minimize cell dialysis allowing 8-pCPT-2′-O-Me-cAMP to reach a higher intracellular concentration. However, in these experiments, 8-pCPT-2′-O-Me-cAMP (30 μM) inhibited bTREK-1 by only 10.2 ± 6.4% (n = 5), while ACTH (200 pM) inhibited bTREK-1 under the same conditions by 83.8 ± 2.8% (n = 7) (Fig. 5, A and C).

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