RT Journal Article SR Electronic T1 Purine and Pyrimidine Nucleotides Inhibit a Noninactivating K+ Current and Depolarize Adrenal Cortical Cells through a G Protein-Coupled Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 364 OP 376 DO 10.1124/mol.55.2.364 VO 55 IS 2 A1 Lin Xu A1 John J. Enyeart YR 1999 UL http://molpharm.aspetjournals.org/content/55/2/364.abstract AB Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that sets the resting membrane potential and may mediate depolarization-dependent cortisol secretion. External ATP stimulates cortisol secretion through activation of a nucleotide receptor. In whole-cell patch clamp recordings from bovine AZF cells, we found that ATP selectively inhibited IACK+ current by a maximum of 75.7 ± 3% (n = 13) with a 50% inhibitory concentration of 1.3 μM. A rapidly inactivating A-type K+ current was not inhibited by ATP. Other nucleotides, including ADP and the pyrimidines UTP and UDP, also inhibited IAC, whereas 2-methylthio-ATP (2-MeSATP) and CTP were completely ineffective. The rank order of potency for six nucleotides was UTP = ADP > ATP > UDP ≫ 2-MeSATP = CTP. At maximally effective concentrations, UTP, ADP, and UDP inhibited IAC current by 81.4 ± 5.2% (n = 7), 70.7 ± 7.2% (n= 4), and 65.2 ± 7.9% (n = 5), respectively. Inhibition of IAC by external ATP was reduced from 71.3 ± 3.2% to 22.8 ± 4.5% (n = 18) by substituting guanosine 5′-O-2-(thio) diphosphate for GTP in the patch pipette. Inhibition of IAC by external ATP (10 μM) was markedly suppressed (to 17.3 ± 5.5%,n = 9) by the nonspecific protein kinase antagonist staurosporine (1 μM) and eliminated by substituting the nonhydrolyzable ATP analog 5-adenylyl-imidodiphosphate or UTP for ATP in the pipette. ATP-mediated inhibition of IAC was not altered by the kinase C antagonist calphostin C, the calmodulin inhibitory peptide, or by buffering the intracellular (pipette) Ca++ with 20 mM 1,2-bis-(2-aminophenoxy)ethane-N, N,N′,N′-tetraacetic acid. In current clamp recordings, ATP and UTP (but not CTP) depolarized AZF cells at concentrations that inhibited IAC K+ current. These results demonstrate that bovine AZF cells express a nucleotide receptor with a P2Y3 agonist profile that is coupled to the inhibition of IAC K+ channels through a GTP-binding protein. The inhibition of IAC K+ current and associated membrane depolarization are the first cellular responses demonstrated to be mediated through this receptor. Nucleotide inhibition of IAC proceeds through a pathway that is independent of phospholipase C, but that requires ATP hydrolysis. The identification of a new signaling pathway in AZF cells, whereby activation of a nucleotide receptor is coupled to membrane depolarization through inhibition of a specific K+ channel, suggests a mechanism for ATP-stimulated corticosteroid secretion that depends on depolarization-dependent Ca++ entry. This may be a means of synchronizing the stress-induced secretion of corticosteroids and catecholamines from the adrenal gland.