Adrenocorticotropic hormone and cAMP inhibit noninactivating K+ current in adrenocortical cells by an A-kinase-independent mechanism requiring ATP hydrolysis.
Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that is inhibited by adrenocorticotropic hormone (ACTH) at picomolar concentrations. Inhibition of IAC may be a critical step in depolarization-dependent Ca2+ entry leading to cortisol secretion. In whole-cell patch clamp recordings from AZF cells, we have characterized properties of IAC and the signalling pathway by which ACTH inhibits this current. IAC was identified as a voltage-gated, outwardly rectifying, K(+)-selective current whose inhibition by ACTH required activation of a pertussis toxin-insensitive GTP binding protein. IAC was selectively inhibited by the cAMP analogue 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (8-pcpt-cAMP) with an IC50 of 160 microM. The adenylate cyclase activator forskolin (2.5 microM) also reduced IAC by 92 +/- 4.7%. Inhibition of IAC by ACTH, 8-pcpt-cAMP and forskolin was not prevented by the cAMP-dependent protein kinase inhibitors H-89 (5 microM), cAMP-dependent protein kinase inhibitor peptide (PKI[5-24]) (2 microM), (Rp)-cAMPS (500 microM), or by the nonspecific protein kinase inhibitor staurosporine (100 nM) applied externally or intracellularly through the patch pipette. At the same concentrations, these kinase inhibitors abolished 8-pcpt-cAMP-stimulated A-kinase activity in AZF cell extracts. In intact AZF cells, 8-pcpt-cAMP activated A-kinase with an EC50 of 77 nM, a concentration 2,000-fold lower than that inhibiting IAC half maximally. The active catalytic subunit of A-kinase applied intracellularly through the recording pipette failed to alter functional expression of IAC. The inhibition of IAC by ACTH and 8-pcpt-cAMP was eliminated by substituting the nonhydrolyzable ATP analogue AMP-PNP for ATP in the pipette solution. Penfluridol, an antagonist of T-type Ca2+ channels inhibited 8-pcpt-cAMP-induced cortisol secretion with an IC50 of 0.33 microM, a concentration that effectively blocks Ca2+ channel in these cells. These results demonstrate that IAC is a K(+)-selective current whose gating is controlled by an unusual combination of metabolic factors and membrane voltage. IAC may be the first example of an ionic current that is inhibited by cAMP through an A-kinase-independent mechanism. The A-kinase-independent inhibition of IAC by ACTH and cAMP through a mechanism requiring ATP hydrolysis appears to be a unique form of channel modulation. These findings suggest a model for cortisol secretion wherein cAMP combines with two separate effectors to activate parallel steroidogenic signalling pathways. These include the traditional A-kinase-dependent signalling cascade and a novel pathway wherein cAMP binding to IAC K+ channels leads to membrane depolarization and Ca2+ entry. The simultaneous activation of A-kinase- and Ca(2+)-dependent pathways produces the full steroidogenic response.
Enyeart
J J
JJ
Department of Pharmacology, Ohio State University, College of Medicine, Columbus 43210-1239, USA. jenyeart@magnus.acs.ohio-state.edu
Mlinar
B
B
Enyeart
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000302:Adrenal Cortex / Q000166:cytology / Q000502:physiology*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology; D015220:Calcium Channels / Q000502:physiology; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000502:physiology; D006854:Hydrocortisone / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D009712:Nucleotides, Cyclic / Q000494:pharmacology; D011188:Potassium / Q000037:antagonists & inhibitors / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology
D015220:Calcium Channels; D009712:Nucleotides, Cyclic; D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D011188:Potassium; D002118:Calcium; D006854:Hydrocortisone