Abstract

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_ACK⁺ 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.