Abstract
Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels whose inhibition by cAMP is coupled to membrane depolarization and cortisol secretion through complex signaling mechanisms. cAMP analogs with substitutions in the 6 position of the adenine ring selectively activate cAMP-dependent protein kinase (PKA) but not exchange proteins activated by cAMP (Epacs). In whole-cell patch-clamp recordings from AZF cells, we found that 6-benzoyl-cAMP (6-Bnz-cAMP) and 6-monobutyryl-cAMP potently inhibit bTREK-1 K+ channels, even under conditions in which PKA activity was abolished. Specifically, when applied through the patch electrode, 6-Bnz-cAMP inhibited bTREK-1 with an IC50 of less than 0.2 μM. Inhibition of bTREK-1 by 6-Bnz-cAMP was not diminished by PKA antagonists, including N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), adenosine 3′-5′cyclic monophosphothiate, Rp-isomer, protein kinase inhibitor (PKI) (6–22) amide, and myristoylated PKI (14–22), applied alone or in combination, externally and intracellularly through the patch pipette. Under similar conditions, these same antagonists completely blocked PKA activation by 6-Bnz-cAMP. Inhibition of bTREK-1 by 6-Bnz-cAMP was voltage-independent and eliminated in the absence of ATP in the pipette solution. 6-Bnz-cAMP also produced delayed increases in cortisol synthesis and the expression of CYP11a1 mRNA that were only partially blocked by PKA antagonists. These results indicate that 6-Bnz-cAMP and other 6-substituted cAMP analogs can inhibit bTREK-1 K+ channels and stimulate delayed increases in cortisol synthesis by AZF cells through a PKA- and Epac-independent mechanism. They also suggest that adrenocorticotropin and cAMP function in these cells through a third cAMP-dependent protein. Finally, although 6-modified cAMP analogs exhibit high selectivity in activating PKA over Epac, they also may interact with other unidentified proteins expressed by eukaryotic cells.
Footnotes
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK47875].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.057075
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ABBREVIATIONS:
- AZF
- adrenal zona fasciculata
- PKI
- protein kinase inhibitor; 6-Bnz-cAMP - 6-benzoyl-cAMP
- 6-MB-cAMP
- 6-monobutyrl-cAMP
- PKA
- cAMP-dependent protein kinase, Epac, exchange proteins activated by cAMP
- CaMK
- calmodulin-dependent protein kinase, BAPTA - 1,2 bis-(2-aminophenoxy)ethane-N,N,N′,N"-tetraacetic acid
- DMEM
- Dulbecco's modified Eagle's medium
- DMEM/F12+
- Dulbecco's modified Eagle's medium/F12 (1:1) containing fetal bovine serum, penicillin, mg/ml streptomycin, and the antioxidants tocopherol, selenite, and ascorbic acid
- FBS
- fetal bovine sera
- H-89
- N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline
- 8CPT-2′-OMe-cAMP
- 8-chlorophenylthio-2′-O-methyl-cAMP
- HEK
- human embryonic kidney
- MAP
- mitogen-activated protein
- PBS
- phosphate-buffered saline
- Rp-cAMPS
- adenosine 3′-5′cyclic monophosphothiate, Rp-isomer
- KN-93
- 2-(N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl))amino-N-(4-chlorocinnamyl)-N-methylbenzylamine
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene.
- Received April 15, 2009.
- Accepted September 4, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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