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ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms.

ABSTRACT: Bovine adrenal zona fasciculata (AZF) cells express Ca(v)3.2 T-type Ca(2+) channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca(v)3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3-6 days markedly enhanced the expression of Ca(v)3.2 current. The increase in Ca(v)3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Ca(v)3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 mum) and 6-benzoyladenosine cAMP (300 mum) induced CACNA1H mRNA, but not Ca(v)3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10-50 mum) enhanced CACNA1H mRNA and Ca(v)3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Ca(v)3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Ca(v)3.2 current and mRNA with a potency and effectiveness similar to the parent compound. The Epac activator 8CPT-2'-O-methyl-cAMP and its metabolites 8CPT-2'-OMe-5'-AMP and 8CPT-2'-O-methyl-adenosine increased CACNA1H mRNA and Ca(v)3.2 current; Sp-8CPT-2'-O-methyl-cAMP increased neither Ca(v)3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca(2+) current. Specifically, ACTH induces expression of CACNA1H mRNA and Ca(v)3.2 current in AZF cells by mechanisms that depend at most only partly on cAMP. In contrast, cAMP enhances expression of CACNA1H mRNA but not the corresponding Ca(2+) current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Ca(v)3.2 current indirectly through metabolites. ACTH and the metabolites may induce Ca(v)3.2 expression by the same, unidentified mechanism.

SUBMITTER: Liu H

PROVIDER: S-EPMC2888416 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms.

Liu Haiyan H Enyeart Judith A JA Enyeart John J JJ

The Journal of biological chemistry 20100427 26

Bovine adrenal zona fasciculata (AZF) cells express Ca(v)3.2 T-type Ca(2+) channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca(v)3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3-6 days markedly enhanced the expression of Ca(v)3.2 current. The increase in Ca(v)3.2 current was preceded by an increase in correspond ...[more]

PMID: 20424171

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Project description: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.

Project description:Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation). However, there is a limited understanding of the underlying internal pacemaker mechanisms involved in the crosstalk between cholinergic receptors and the decrease in the SANC AP firing rate (i.e., negative chronotropic modulation). We hypothesize that changes in AC-cAMP/PKA activity are crucial for mediating either decrease or increase in the AP firing rate and that the change in rate is due to both internal and membrane mechanisms. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, PKA activity and AP firing rate were tightly linked in response to either adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic stimulation (by carbachol, CCh). To identify the main molecular targets that mediate between PKA signaling and pacemaker function, we developed a mechanistic computational model. The model includes a description of autonomic-nervous receptors, post- translation signaling cascades, membrane molecules, and internal pacemaker mechanisms. Yielding results similar to those of the experiments, the model simulations faithfully reproduce the changes in AP firing rate in response to CCh or ISO or a combination of both (i.e., accentuated antagonism). Eliminating AC-cAMP-PKA signaling abolished the core effect of autonomic receptor stimulation on the AP firing rate. Specifically, disabling the phospholamban modulation of the SERCA activity resulted in a significantly reduced effect of CCh and a failure to increase the AP firing rate under ISO stimulation. Directly activating internal pacemaker mechanisms led to a similar extent of changes in the AP firing rate with respect to brain receptor stimulation. Thus, Ca2+ and cAMP/PKA-dependent phosphorylation limits the rate and magnitude of chronotropic changes in the spontaneous AP firing rate.

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ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms.

Publications

ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms.

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