ADRENOCORTICOTROPIC HORMONE AND CAMP INHIBIT NONINACTIVATING K-CELLS BY AN A-KINASE-INDEPENDENT MECHANISM REQUIRING ATP HYDROLYSIS( CURRENTIN ADRENOCORTICAL)

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (I-AC) that is inhibited by adrenocorticotropic hormone (AC TH) at picomolar concentrations. Inhibition of I-AC may be a critical step in depolarization-dependent Ca2+ entry leading to cortisol secret ion. In whole-cell patch clamp recordings from AZF cells, we have char acterized properties of I-AC and the signalling pathway by which ACTH inhibits this current. I-AC was identified as a voltage-gated, outward ly rectifying, K+-selective current whose inhibition by ACTH required activation of a pertussis toxin-insensitive GTP binding protein. I-AC was selectively inhibited by the cAMP analogue 8-(4-chlorophenylthio)- adenosine 3':5'-cyclic monophosphate (8-pcpt-cAMP) with an IC50 of 160 mu M. The adenylate cyclase activator forskolin (2.5 mu M also reduce d I-AC by 92 +/- 4.7%. Inhibition of I-AC by ACTH, 8-pcpt-cAMP and for skolin was not prevented by the cAMP-dependent protein kinase inhibito rs H-89 (5 mu M), cAMP-dependent protein kinase inhibitor peptide (PKI [5-24]) (2 mu M), (Rp)-cAMPS (500 mu M), or by the nonspecific protein kinase inhibitor staurosporine (100 nM) applied externally or intrace llularly through the patch pipette. At the same concentrations, these kinase inhibitors abolished 8-pcpt-cAMP-stimulated A-kinase activity i n AZF cell extracts. In intact AZF cells, 8-pcpt-cAMP activated A-kina se with an EC(50) of 77 nM, a concentration 2,000-fold lower than that inhibiting I-AC: half maximally. The active catalytic subunit of A-ki nase applied intracellularly through the recording pipette failed to a lter functional expression of I-AC. The inhibition of I-AC by ACTH and 8-pcpt-cAMP was eliminated by substituting the nonhydrolyzable ATP an alogue AMP-PNP for ATP in the pipette solution. Penfluridol, an antago nist of T-type Ca2+ channels inhibited 8-pcpt-cAMP-induced cortisol se cretion with an IC50 of 0.33 mu M, a concentration that effectively bl ocks Ca2+ channel in these cells. These results demonstrate that I-AC is a K+-selective current whose gating is controlled by an unusual com bination of metabolic factors and membrane voltage. I-AC may be the fi rst example of an ionic current that is inhibited by cAMP through an A -kinase-independent mechanism. The A-kinase-independent inhibition of I-AC: by ACTH and cAMP through a mechanism requiring ATP hydrolysis ap pears to be a unique for-m of channel modulation. These findings sugge st a model for cortisol secretion wherein cAMP combines with two separ ate effectors to activate parallel steroidogenic signalling pathways. These include the traditional A-kinase-dependent signalling cascade an d a novel pathway wherein cAMP binding to I-AC K+ channels leads to me mbrane depolarization and Ca2+ entry. The simultaneous activation of A -kinase- and Ca2+-dependent pathways produces the full steroidogenic r esponse.