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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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Concentration-dependent inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP. K+ currents were recorded from AZF cells with standard pipette solution or the same solution supplemented with 8-pCPT-2′-O-Me-cAMP (EA) at concentrations from 1 to 30 μM. Currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of −80 mV with and without depolarizing prepulses. (A–C) Time-dependent increase in bTREK-1 and inhibition by 8-pCPT-2′-O-Me-cAMP (EA). Current traces recorded with (right) and without (left) depolarizing prepulses at indicated times. bTREK-1 amplitudes are plotted at right. Open circles indicate traces recorded with depolarizing prepulse. (D) Summary of experiments as in A–C. Bars indicate bTREK-1 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations. (E) Effect of 8-pCPT-2′-O-Me-cAMP (EA) on Kv1.4 current. Bars indicate Kv1.4 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations in control saline and in the presence of 8-pCPT-2′-O-Me-cAMP (30 μM) (EA).
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fig6: Concentration-dependent inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP. K+ currents were recorded from AZF cells with standard pipette solution or the same solution supplemented with 8-pCPT-2′-O-Me-cAMP (EA) at concentrations from 1 to 30 μM. Currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of −80 mV with and without depolarizing prepulses. (A–C) Time-dependent increase in bTREK-1 and inhibition by 8-pCPT-2′-O-Me-cAMP (EA). Current traces recorded with (right) and without (left) depolarizing prepulses at indicated times. bTREK-1 amplitudes are plotted at right. Open circles indicate traces recorded with depolarizing prepulse. (D) Summary of experiments as in A–C. Bars indicate bTREK-1 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations. (E) Effect of 8-pCPT-2′-O-Me-cAMP (EA) on Kv1.4 current. Bars indicate Kv1.4 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations in control saline and in the presence of 8-pCPT-2′-O-Me-cAMP (30 μM) (EA).

Mentions: Because of the limited effectiveness of the cAMP analogues when applied extracellularly at low concentrations and the associated uncertainty of their intracellular concentrations, we measured the effects of 8-pCPT-2′-O-Me-cAMP on bTREK-1 K+ channel activity when it was applied directly to the cytoplasm through the pipette. When applied through the pipette at concentrations from 1 to 30 μM, 8-pCPT-2′-O-Me-cAMP potently and effectively suppressed the time-dependent growth of bTREK-1 with an IC50 of 0.63 μM (Fig. 6, A–D). Notably, at a concentration of 1 μM, 8-pCPT-2′-O-Me-cAMP reduced bTREK-1 current density by 63.7% from a control value of 45.2 ± 5.9 pA/pF (n = 15) to 16.4 ± 4.4 pA/pF (n = 6). Using this low concentration of 8-pCPT-2′-O-Me-cAMP in the pipette, bTREK-1 amplitude initially grew, but then declined to a steady-state value (Fig. 6 B). At higher concentrations, the initial increase in bTREK-1 amplitude was absent and inhibition of bTREK-1 activity was nearly complete (Fig. 6, C and D). The inhibition of bTREK-1 expression by 8-pCPT-2′-O-Me-cAMP was specific. This agent did not alter the expression of the voltage-gated Kv1.4 current in these cells (Fig. 6 E).


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

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

Concentration-dependent inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP. K+ currents were recorded from AZF cells with standard pipette solution or the same solution supplemented with 8-pCPT-2′-O-Me-cAMP (EA) at concentrations from 1 to 30 μM. Currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of −80 mV with and without depolarizing prepulses. (A–C) Time-dependent increase in bTREK-1 and inhibition by 8-pCPT-2′-O-Me-cAMP (EA). Current traces recorded with (right) and without (left) depolarizing prepulses at indicated times. bTREK-1 amplitudes are plotted at right. Open circles indicate traces recorded with depolarizing prepulse. (D) Summary of experiments as in A–C. Bars indicate bTREK-1 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations. (E) Effect of 8-pCPT-2′-O-Me-cAMP (EA) on Kv1.4 current. Bars indicate Kv1.4 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations in control saline and in the presence of 8-pCPT-2′-O-Me-cAMP (30 μM) (EA).
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fig6: Concentration-dependent inhibition of bTREK-1 by 8-pCPT-2′-O-Me-cAMP. K+ currents were recorded from AZF cells with standard pipette solution or the same solution supplemented with 8-pCPT-2′-O-Me-cAMP (EA) at concentrations from 1 to 30 μM. Currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of −80 mV with and without depolarizing prepulses. (A–C) Time-dependent increase in bTREK-1 and inhibition by 8-pCPT-2′-O-Me-cAMP (EA). Current traces recorded with (right) and without (left) depolarizing prepulses at indicated times. bTREK-1 amplitudes are plotted at right. Open circles indicate traces recorded with depolarizing prepulse. (D) Summary of experiments as in A–C. Bars indicate bTREK-1 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations. (E) Effect of 8-pCPT-2′-O-Me-cAMP (EA) on Kv1.4 current. Bars indicate Kv1.4 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations in control saline and in the presence of 8-pCPT-2′-O-Me-cAMP (30 μM) (EA).
Mentions: Because of the limited effectiveness of the cAMP analogues when applied extracellularly at low concentrations and the associated uncertainty of their intracellular concentrations, we measured the effects of 8-pCPT-2′-O-Me-cAMP on bTREK-1 K+ channel activity when it was applied directly to the cytoplasm through the pipette. When applied through the pipette at concentrations from 1 to 30 μM, 8-pCPT-2′-O-Me-cAMP potently and effectively suppressed the time-dependent growth of bTREK-1 with an IC50 of 0.63 μM (Fig. 6, A–D). Notably, at a concentration of 1 μM, 8-pCPT-2′-O-Me-cAMP reduced bTREK-1 current density by 63.7% from a control value of 45.2 ± 5.9 pA/pF (n = 15) to 16.4 ± 4.4 pA/pF (n = 6). Using this low concentration of 8-pCPT-2′-O-Me-cAMP in the pipette, bTREK-1 amplitude initially grew, but then declined to a steady-state value (Fig. 6 B). At higher concentrations, the initial increase in bTREK-1 amplitude was absent and inhibition of bTREK-1 activity was nearly complete (Fig. 6, C and D). The inhibition of bTREK-1 expression by 8-pCPT-2′-O-Me-cAMP was specific. This agent did not alter the expression of the voltage-gated Kv1.4 current in these cells (Fig. 6 E).

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