ACTH Induces Cav3.2 Current and mRNA by cAMP-dependent and cAMP-independent Mechanisms

Haiyan Liu,Judith A Enyeart,John J Enyeart

doi:10.1074/jbc.m110.104190

Haiyan Liu, Judith A Enyeart + Show 1 more

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https://doi.org/10.1074/jbc.m110.104190

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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.

Highlights

We previously found that cAMP inhibits the activity of bTREK-1 Kϩ channels by a mechanism that is independent of PKA but mimicked by Epacspecific cAMP analogs (ESCAs) [27, 28]
We examined the regulation of CACNA1H mRNA and corresponding Cav3.2 Ca2ϩ currents in adrenal zona fasciculata (AZF) cells by adrenocorticotropic hormone (ACTH), cAMP analogs, and their metabolites using Northern blot and patch clamp techniques
At concentrations of 50 and 100 nM, NPS-ACTH increased Cav3.2 current density by an amount similar to that produced by ACTH (10 nM) at 48 and 72 h serum-supplemented media, the Cav3.2 current disappeared (Fig. 2, A and C)

Summary

Introduction

We examined the regulation of CACNA1H mRNA and corresponding Cav3.2 Ca2ϩ currents in AZF cells by ACTH, cAMP analogs, and their metabolites using Northern blot and patch clamp techniques. That ACTH and metabolites of chlorophenylthio-cAMP analogs induce the expression of CACNA1H mRNA and Ca2ϩ current. CACNA1H mRNA Expression—In bovine AZF cells, ACTH enhanced the expression of a Ni2ϩ-sensitive T-type Ca2ϩ current presumed to be Cav3.2.

Results

Conclusion