Vol. 55, Issue 2, 364-376, February 1999

Purine and Pyrimidine Nucleotides Inhibit a Noninactivating K+ Current and Depolarize Adrenal Cortical Cells through a G Protein-Coupled Receptor

Lin Xu and John J. Enyeart

Department of Pharmacology (L.X., J.J.E.) and Neuroscience Program (J.J.E.), The Ohio State University College of Medicine, Columbus, Ohio

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K⁺ current (I_AC) 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 I_AC K⁺ 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 I_AC, 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 I_AC current by 81.4 ± 5.2% (n = 7), 70.7 ± 7.2% (n = 4), and 65.2 ± 7.9% (n = 5), respectively. Inhibition of I_AC 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 I_AC 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 I_AC 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 I_AC 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 I_AC K⁺ channels through a GTP-binding protein. The inhibition of I_AC K⁺ current and associated membrane depolarization are the first cellular responses demonstrated to be mediated through this receptor. Nucleotide inhibition of I_AC 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.