Adrenocorticotropic hormone and cAMP inhibit noninactivating K+ current in adrenocortical cells by an A-kinase-independent mechanism requiring ATP hydrolysis

被引:47
作者
Enyeart, JJ [1 ]
Mlinar, B [1 ]
Enyeart, JA [1 ]
机构
[1] OHIO STATE UNIV,PROGRAM NEUROSCI,COLL MED,COLUMBUS,OH 43210
关键词
potassium channel; adrenal cortex; cortisol; protein kinase;
D O I
10.1085/jgp.108.4.251
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (I-AC) that is inhibited by adrenocorticotropic hormone (ACTH) at picomolar concentrations. Inhibition of I-AC 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 I-AC and the signalling pathway by which ACTH inhibits this current. I-AC 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. 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 reduced I-AC by 92 +/- 4.7%. Inhibition of I-AC by ACTH, 8-pcpt-cAMP and forskolin was not prevented by the cAMP-dependent protein kinase inhibitors 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 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 EC(50) of 77 nM, a concentration 2,000-fold lower than that inhibiting I-AC: half maximally. The active catalytic subunit of A-kinase applied intracellularly through the recording pipette failed to alter functional expression of I-AC. The inhibition of I-AC 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 mu M, a concentration that effectively blocks Ca2+ channel in these cells. These results demonstrate that I-AC is a K+-selective current whose gating is controlled by an unusual combination of metabolic factors and membrane voltage. I-AC 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 I-AC: by ACTH and cAMP through a mechanism requiring ATP hydrolysis appears to be a unique for-m 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 I-AC K+ channels leads to membrane depolarization and Ca2+ entry. The simultaneous activation of A-kinase- and Ca2+-dependent pathways produces the full steroidogenic response.
引用
收藏
页码:251 / 264
页数:14
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