Abstract
bTREK-1 K+ channels set the resting membrane potential of bovine adrenal zona fasciculata (AZF) cells and function pivotally in the physiology of cortisol secretion. Adrenocorticotropic hormone (ACTH) controls the function and expression of bTREK-1 channels through signaling mechanisms that may involve cAMP and downstream effectors including PKA and Epac2. Using patch-clamp and Northern blot analysis, we explored the regulation of bTREK-1 mRNA and K+ current expression by cAMP-analogs and several of their putative metabolites in bovine AZF cells. At concentrations sufficient to activate both PKA and Epac2, 8-Br-cAMP enhanced the expression of both bTREK-1 mRNA and K+ current. 6-Bnz-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K+ current measured at times from 24-96 h. An Epac-selective cAMP analog 8CPT-2'-OMe-cAMP potently stimulated bTREK-1 mRNA and K+ current expression, while the non-hydrolyzable Epac-activator Sp-8CPT-2'-OMe-cAMP was ineffective. Metabolites of 8CPT-2'-OMe-cAMP including 8CPT-2'-OMe-5'AMP and 8CPT-2'-OMe-adenosine promoted the expression of bTREK-1 transcripts and ion current with a temporal pattern, potency, and effectiveness resembling that of the parent compound. Similarly, at low concentrations, 8CPT-cAMP (30 μM), but not its non-hydrolyzable analog Sp-8CPT-cAMP, enhanced the expression of bTREK-1 mRNA and current. 8CPT-cAMP metabolites, including 8CPT-adenosine and 8CPT-adenine, also increased bTREK-1 expression. These results indicate that cAMP increases the expression of bTREK-1 mRNA and K+ current through a cAMP-dependent, but Epac2-independent mechanism. They further demonstrate that one or more metabolites of 8-(4-chlorophenylthio)-cAMP analogs potently stimulate bTREK-1 expression by activation of a novel cAMP-independent mechanism. These findings raise significant questions regarding the specificity of 8-(4-chlorophenylthio)-cAMP-analogs as cAMP-mimetics.
- ACTH
- Purinergic
- cAMP
- Protein Kinase A
- Regulation of gene expression
- Regulation - transcriptional
- Endocrine cells
Footnotes
- Received October 22, 2009.
- Revision received December 21, 2009.
- Accepted December 21, 2009.
- The American Society for Pharmacology and Experimental Therapeutics