Introduction

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Extracellular ATP acts as a neurotransmitter or local hormone to elicit responses in a variety of cells. By activation of specific receptors, ATP mediates or modulates a range of physiological processes that include neurotransmission, contraction of smooth and cardiac muscle cells, inflammatory and immune responses, and secretion of hormones including insulin, glucagon, catecholamines, and corticosteroid hormones (Dalziel and Westfall, 1994; Fredholm et al., 1994; Hoey et al., 1994; Williams and Burnstock, 1997).

Membrane receptors for ATP and other nucleotides can be grouped into two major classes. The P2X receptors form ligand-gated, nonselective cation channels, whereas P2Y receptors are G protein-coupled, membrane proteins (Dubyak and El-Moatassim, 1993; Dalziel and Westfall, 1994; Williams and Burnstock, 1997). Within each of these two major groups, multiple subtypes exist that can be distinguished based on their sensitivity to ATP and other nucleotides. In particular, G protein-coupled P2Y₁ receptors include those that are preferentially activated by purines such as ATP and ADP, and the pyrimidines UTP and UDP (Dubyak and El-Moatassim, 1993; Dalziel and Westfall, 1994; Williams and Burnstock, 1997). The discovery that P2Y receptors included a subtype that was preferentially activated by both purines and pyrimidines led to the subclassification of this family into P2Y₁ and P2Y₂ receptors (Lustig et al., 1993; Parr et al., 1994; Williams and Burnstock, 1997). The cloning of additional P2Y receptors with distinct structures and pharmacological profiles has resulted in the addition of at least six new subtypes to this family (P2Y₃-P2Y₈) (reviewed in Filtz et al., 1997 and Williams and Burnstock, 1997).

In bovine adrenal zona fasciculata (AZF) cells, ATP, UTP, and ADP stimulate cortisol secretion at concentrations between 10⁶ and 10⁴ M through a Ca⁺⁺-dependent process (Niitsu, 1992; Hoey et al., 1994). The cellular mechanisms by which ATP and other nucleotides stimulate cortisol secretion are unknown. AZF cells express a novel K⁺ channel (I_AC) that appears to set the resting membrane potential (Mlinar et al., 1993a; Enyeart et al., 1996). Corticotropin [adrencorticotropic hormone (ACTH)] and AII, two peptide hormones that physiologically regulate cortisol secretion, inhibit I_AC and depolarize AZF cells at concentrations identical with those that stimulate steroidogenesis (Mlinar et al., 1993a). I_AC channels couple peptide receptor activation to membrane depolarization, Ca⁺⁺ entry, and cortisol secretion (Enyeart et al., 1993; Mlinar et al., 1993a). To determine whether nucleotide receptors on AZF cells might also be linked to I_AC inhibition, we have studied the effect of ATP and other nucleotides on I_AC current in whole-cell patch clamp recordings from bovine AZF cells.

	Materials and Methods

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Tissue culture media, antibiotics, fibronectin, and fetal bovine sera were obtained from Gibco (Grand Island, NY). Coverslips were purchased from Bellco Glass, Inc. (Vineland, NJ). Enzymes, ACTH(1-24), MgATP, NaATP, NaUTP, NaUDP, KADP, 5-adenylyl-imidodiphosphate (AMP-PNP, lithium salt), NaCTP, NaGTP, guanosine 5'-O-2-(thio)-diphosphate (GDP--S), 2-MeSATP, 1,2-bis-(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (BAPTA), and staurosporine were obtained from Sigma Chemical Company (St. Louis, MO). Calmodulin inhibitory peptide (residues 290-309 of CaM kinase II) was obtained from Biomol (Plymouth Meeting, PA).

Isolation and Culture of Adrenocortical Cells. Bovine adrenal glands were obtained from steers (age range, 1-3 years) within 60 min of slaughter at a local slaughterhouse. Fatty tissue was removed immediately and the glands were transported to the laboratory in ice-cold phosphate-buffered saline (PBS) containing 0.2% dextrose. Isolated AZF cells were prepared as described previously (Gospodarowicz et al., 1977) with some modifications. In a sterile tissue culture hood, the adrenals were cut in half lengthwise and the lighter medulla tissue was trimmed away from the cortex and discarded. The capsule with attached glomerulosa, and thicker fasciculata layer were then dissected into pieces approximately 1.0 × 1.0 × 0.5 cm. A Stadie-Riggs tissue slicer (Thomas Scientific) was used to separate fasciculata tissue from the glomerulosa layers by slicing 0.3- to 0.5-mm slices from the larger pieces. The first medulla/fasciculata slices were discarded. One to two subsequent fasciculata slices were saved in cold, sterile PBS/0.2% dextrose. The fasciculata/glomerulosa margin (about 0.5 mm) and capsule with attached glomerulosa were discarded. Fasciculata tissue slices were then diced into 0.5-mm³ pieces and dissociated with 2 mg/ml (about 200-300 U/ml) of Type I collagenase (neutral protease activity not exceeding 100 units/mg of solid), 0.2 mg/ml deoxyribonuclease in Dulbecco's modified Eagle's medium (DMEM)/F12 for approximately 1 h at 37°C, triturating after 30 and 45 min with a sterile, plastic transfer pipette. The tissue/cell suspension was filtered through two layers of sterile cheesecloth and then centrifuged to pellet cells at 100g for 5 min. Undigested tissue remaining in the cheesecloth was collagenase-treated for an additional hour. Pelleted cells were washed with DMEM/0.2% BSA, centrifuging as before. After resuspension in DMEM, cells were filtered through 200-µm stainless steel mesh to remove clumps. Dispersed cells were again centrifuged and either resuspended in DMEM/F12 (1:1) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin and plated for immediate use, or resuspended in FBS/5% DMSO, divided into 1-ml aliquots, each containing about 4 × 10⁶ cells, and stored in liquid nitrogen for future use. Cells were plated in 35-mm dishes containing 9-mm² glass coverslips that had been treated with fibronectin (10 µg/ml) at 37°C for 30 min and then rinsed with warm, sterile PBS immediately before adding cells. Dishes were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Patch Clamp Experiments. Patch clamp recordings of K⁺ channel currents were made in the whole-cell configuration. The standard pipette solution was 120 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES, 11 mM BAPTA, and 200 µM GTP, and 5 mM MgATP with pH buffered to 7.2 using KOH. Deviations from the standard solution are described in the text. Pipette [Ca⁺⁺] was determined using the "Bound and Determined" program (Brooks and Storey, 1992). The external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl, 2 mM MgCl₂, 10 mM HEPES, and 5 mM glucose, pH 7.4, using NaOH. All solutions were filtered through 0.22-µm cellulose acetate filters. The Na⁺ salts of ATP, UTP, and CTP were applied externally by bath perfusion controlled manually by a six-way rotary valve.

	Results

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Bovine AZF cells express two types of K⁺ current, a rapidly inactivating, voltage-gated A-type K⁺ current, and a noninactivating weakly voltage-dependent current (I_AC). I_AC is present initially, but grows dramatically over a period of minutes in whole-cell recordings, provided that ATP or other nucleotides are present at millimolar concentrations in the recording pipette (Mlinar and Enyeart, 1993; Mlinar et al., 1993a; Enyeart et al., 1996; Enyeart et al., 1997). The absence of time and voltage-dependent inactivation of the I_AC K⁺ current allowed it to be easily isolated for measurement in whole-cell recordings using either of two voltage clamp protocols. When voltage steps of 300-ms duration are applied from a holding potential of 80 mV to a test potential of +20 mV, I_AC could be measured selectively near the end of a voltage step where the rapidly inactivating A-type current had completely inactivated (Fig. 1A, left traces). Alternatively, I_AC was selectively activated with an identical voltage step, after a 10-s prepulse to 20 mV had fully inactivated the A-type K⁺ current (Fig. 1A, right traces). Results reported in this study were obtained in recordings from more than 200 AZF cells.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Time- and concentration-dependent inhibition of IAC by external ATP. Whole-cell K+ currents were recorded from bovine AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without 10-s prepulses to 20 mV, which inactivated A-type K+ current. After IAC reached a stable amplitude, cells were superfused with NaATP at various concentrations. A, K+ current records made with patch pipettes containing standard pipette solution containing 5 mM MgATP and 200 µM GTP, with (right) or without (left) 10-s prepulses to 20 mV. Numbers correspond to currents immediately after initiating whole-cell recording (1), after IAC had reached a maximum amplitude (2), and after inhibition by ATP (10 µM) (3). B, IAC amplitudes recorded with (open circles) or without (solid circles) depolarizing prepulses are plotted against time. NaATP (10 µM) was perfused as indicated. C, inhibition curve. Fraction of unblocked IAC is plotted against ATP concentration. Data were fit with an equation of the form: I/Imax = 1/[1+(B/Kd)x], where B is NaATP concentration, Kd is the equilibrium dissociation constant, and x is the Hill coefficient.

Selective Inhibition of I_AC by External Nucleotides. ATP applied to AZF cells externally by bath perfusionproduced a selective, concentration-dependent inhibition of I_AC (Fig. 1). In these experiments, I_AC was recorded at 30-s intervals. When this current reached a stable maximum amplitude, ATP was superfused at concentrations ranging from 0.1 to 100 µM. Inhibition of I_AC by ATP began after a delay of 1.5 to 3 min and required several additional minutes to reach a steady-state value (Fig. 1B). ATP inhibited I_AC half-maximally with an estimated 50% inhibitory concentration (IC₅₀) of 1.28 µM. Even at maximally effective concentrations, ATP did not completely inhibit I_AC, reducing this current by 71.3 ± 3.2% (n = 26) and 73.9 ± 3.5% (n = 13) at concentrations of 10 and 100 µM, respectively (Fig. 1C). Inhibition of I_AC by ATP was, in general, poorly reversible even with prolonged washing (Fig. 1B). In contrast to I_AC current, the rapidly inactivating voltage-gated A-type current was not reduced by ATP (Fig. 1A).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Voltage-independent inhibition of IAC by ATP. K+ currents were activated at 30-s intervals by voltage steps of varying size from a holding potential of 80 mV before and after superfusing the cell with 10 µM NaATP. A, current records at test potentials between 60 and +60 mV (in 10-mV increments) before and after superfusing ATP, as indicated. B, current-voltage relationships. IAC current amplitudes from experiment shown in A are plotted against test voltage.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Inhibition of IAC K+ current by purines and pyrimidines. A, AZF cells were clamped in whole-cell configuration and voltage steps were applied at 30-s intervals from a holding potential of 80 mV with (middle) or without (top) 10-s prepulses to 20 mV. After IAC reached a maximum amplitude, cells were superfused with UTP, ADP, UDP, 2-MeSATP, or CTP each at a concentration of 10 µM or 200 pM ACTH, as indicated. Current traces and corresponding plots of IAC amplitude against time before and after superfusion of nucleotides for four representative cells. B, summary of results from experiments such as in A. Bars indicate fraction of IAC remaining after steady-state block by each nucleotide as indicated. Values are mean ± S.E.M. for the indicated number of cells. C, inhibition curves for 2-MeSATP, ADP, and UTP. Fraction of IAC remaining is plotted against nucleotide concentration. Data were fit with an equation of the same form as in Fig. 1. N values range from 3 to 7 for each concentration.

Inhibition of I_AC by ATP Involves a G Protein-Coupled Receptor. When AZF cells were voltage clamped at the standardholding potential of 80 mV, superfusion of ATP never elicited an inward current, as would have occurred if an ATP-gated nonselective cation channel were activated. This finding, coupled with the observed order of effectiveness of nucleotides as inhibitors of I_AC current, suggested that these activated a G protein-coupled nucleotide receptor of the P2Y family (Filtz et al., 1997; Williams and Burnstock, 1997). To determine whether ATP-mediated inhibition of I_AC required activation of a G protein, GTP in the pipette solution was replaced with the inactive guanine nucleotide GDP--S. With 1 mM GDP--S in the patch pipette, ATP (10 µM) inhibited I_AC by only 22.8 ± 4.5% (n = 18), compared with 71.3 ± 3.2% (n = 26) under control conditions (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of guanine nucleotides on ATP-mediated IAC inhibition. IAC was measured at 30-s intervals as described in the legend of Fig. 1 with standard pipette solution (200 µM GTP) or the same solution containing 1 mM GDP--S in place of GTP. After IAC reached a stable value, cells were superfused with 10 µM NaATP. A, K+ currents were recorded in the presence of GDP--S with (right) and without (left) 10-s prepulses to 20 mV immediately after initiating whole-cell recording (1), after IAC had reached a maximum value (2), and after steady-state inhibition by 10 µM ATP (3). B, IAC amplitudes recorded using pipette solution containing GDP--S either with (open circles) or without (solid circles) depolarizing pulses are plotted against time. C, summary of results from experiments as in A or B. Bars indicate fraction of IAC remaining after steady-state block by 10 µM ATP with 200 µM GTP or 1 mM GDP--S in pipette as indicated. Results are mean ± S.E.M. of indicated number of cells.

I_AC Inhibition by ATP Is Independent of Phospholipase C (PLC)-Generated Second Messengers. Experiments with GDP--S indicated that ATP-inhibited I_AC through a G protein-coupled receptor. In many cells, the binding of ATP to G protein-dependent nucleotide receptors is coupled to activation of PLC (Dubyak and El-Moatassim, 1993). PLC-catalyzed cleavage of phosphatidyl inositol 4,5-bisphosphate generates second messengers, including diacylglycerol and IP₃, which, respectively, activate protein kinase C and release intracellular Ca⁺⁺. We have explored the possibility that ATP-mediated inhibition of I_AC occurs through a PLC-activated pathway.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   IAC inhibition by ATP is independent of kinase C and Ca++/calmodulin. K+ currents were recorded from AZF cells using the two protocols described in the legend of Fig. 1. Pipettes contained standard internal solution each supplemented with 500 nM calphostin C (A) or 2.5 µM CaM Kinase inhibitory peptide and 20 mM BAPTA (B). After IAC reached a stable maximum value, cells were superfused with 10 µM NaATP. A and B, calphostin C and calmodulin inhibitory peptide. Current traces recorded with (middle) and without (left) 10-s prepulses to 20 mV immediately after initiating whole-cell recording (1), after IAC had reached a maximum value (2), and after block by 10 µM ATP. IAC amplitudes recorded either with (open circles) or without (solid circles) 10-s prepulses to 20 mV are plotted against time at right. C, summary of results from experiments as in A and B. Bars indicate fraction of IAC remaining after block by 10 µM ATP with pipette solutions containing calphostin or calmodulin inhibitory peptide and 20 mM BAPTA as indicated. Results are mean ± S.E.M. of indicated number of experiments.

Effect of Staurosporine and AMP-PNP on I_AC Inhibition by ATP. Staurosporine is a potent nonselective protein kinase antagonist. This microbial alkaloid inhibits most serine/threonine protein kinases, including A kinase, C kinase, and Ca⁺⁺/CaM kinase II with IC₅₀ values of <20 nM (Tamaoki, 1991). As illustrated in Fig. 6A, staurosporine (1 µM) applied intracellularly through the pipette solution markedly reduced the inhibition of I_AC K⁺ current by 10 µM ATP. In this experiment, ATP inhibited I_AC by less than 5%. In contrast ACTH (100 pM), which functions through a staurosporine-insensitive mechanism (Enyeart et al., 1996), inhibits I_AC almost completely. In a total of nine similar experiments, ATP (10 µM) inhibited I_AC by only 17.3 ± 5.5% when the pipette solution contained 1 µM staurosporine (Fig. 6C), compared with the control value of 71.3 ± 3.2% (n = 26).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Inhibition of IAC by ATP is suppressed by staurosporine and AMP-PNP. K+ currents were recorded in AZF cells using the two protocols described in the legend of Fig. 1. Patch pipettes contained standard solution supplemented with 1 µM staurosporine (A) or the same solution in which AMP-PNP (1 mM) was substituted for MgATP (B). After IAC reached a stable amplitude, cells were superfused with 10 µM NaATP. A and B, current traces recorded with (middle) and without (left) 10-s prepulses to 20 mV immediately after initiating whole-cell recording (1), after IAC had reached a maximum value (2), after block by 10 µM ATP (3), and after block by 100 pM ACTH (4). IAC amplitudes recorded either with (open circles) or without (solid circles) 10-s prepulses to 20 mV are plotted against time at right. C, summary of results from experiments as in A and B. Bars indicate fraction of IAC remaining after block by 10 µM external ATP with standard pipette solution or the same solution supplemented with staurosporine or one in which AMP-PNP was substituted for MgATP.

Effect of Intracellular UTP on I_AC Inhibition by External ATP. In addition to intracellular ATP, other nucleotides including UTP can, when added to the pipette at millimolar concentrations, activate I_AC K⁺ channels (Enyeart et al., 1997). Although UTP can bind to an intracellular site to activate I_AC K⁺ channels, it does not replace ATP as a substrate for most kinases or ATPases (Glynn and Hoffman, 1971; Lemaire et al., 1974). When 5 mM UTP was substituted for ATP in the pipette solution, externally applied ATP (10 µM) failed to inhibit I_AC channels. In five experiments in which the pipette solution contained only 5 mM UTP and no ATP, external ATP (10 µM) inhibited I_AC by only 2.0 ± 1.7% (Fig. 7). As previously reported, ACTH (100 pM) also failed to inhibit I_AC under these conditions (Enyeart et al., 1997).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Inhibition of IAC by external ATP requires intracellular ATP. K+ currents were recorded with the two voltage protocols described in the legend of Fig. 1. Patch pipettes contained standard solution supplemented with 5 mM NaUTP, either alone or in combination with MgATP at concentrations of 50 µM, 200 µM, 2 mM, or 5 mM, as indicated. After IAC reached a maximum value, cells were superfused sequentially with ATP (10 µM) and ACTH (100 pM). A, traces show currents recorded with (bottom) and without (top) 10-s depolarizing prepulses to 20 mV immediately after initiating recording (1), after IAC had reached a maximum value (2), after superfusing 10 µM NaATP (3), and after superfusing 100 pM ACTH (4). Pipette ATP and UTP concentrations are as indicated. IAC amplitudes recorded either with (open circles) or without (solid circles) depolarizing prepulses are plotted beneath corresponding traces. B, summary of results of experiments as in A. Bars indicate fraction of IAC remaining after inhibition by 10 µM external NaATP with pipette solutions containing ATP and UTP at the indicated concentrations. Results are mean ± S.E.M. of the indicated number of determinations.

Effect of Nucleotides on Membrane Potential. In previous studies, we have shown that I_AC K⁺ channels display little voltage dependence and remain open at negative membrane potentials (Mlinar et al., 1993a; Enyeart et al., 1996), characteristics indicative of a channel that sets the resting potential. These results suggest that ATP and other nucleotides that inhibit I_AC, when applied externally, also should depolarize AZF cells.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of ATP and UTP on IAC K+ current and membrane potential. K+ currents were recorded in the whole-cell mode using the two voltage clamp protocols described in the legend of Fig. 1. When IAC reached a stable maximum value, membrane potential was recorded upon switching to current clamp and cells were superfused with either 10 µM NaATP (A) or NaUTP (B). K+ currents in A and B were recorded with (bottom traces) and without (top traces) 10-s depolarizing prepulses to 20 mV immediately after initiating whole-cell recording (1), after IAC had reached a maximum value (2), and after inhibition with ATP (left) or UTP (right) (3). Bottom panels show membrane potential recordings before, during, and after superfusing ATP or UTP as indicated.

	Discussion

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Bovine AZF cells express a nucleotide receptor that, when activated by ATP, ADP, UTP, and UDP, inhibits I_AC K⁺ current. The order of nucleotide potency and effectiveness in inhibiting I_AC indicates that this receptor is distinct from purine-specific P2Y₁ and P2Y₂ receptors but similar to P2Y₃ receptors (Lustig et al., 1993; Parr et al., 1994; Webb et al., 1996; Filtz et al., 1997; Williams and Burnstock, 1997). Members of the P2Y family of receptors are coupled through a G protein-to-PLC activation. However, I_AC inhibition by ATP appears to occur through a distinct mechanism involving a staurosporine-sensitive kinase and, possibly, an ATPase. The inhibition of I_AC K⁺ current by nucleotides was tightly correlated with their effectiveness in depolarizing AZF cells. ATP and UTP inhibit I_AC current and depolarized AZF cells at nearly identical concentrations. 2-MeSATP and CTP were ineffective in both respects.

Overall, purine and pyrimidine nucleotides inhibit I_AC and depolarize AZF cells at concentrations identical with those that stimulate cortisol secretion (Hoey et al., 1994). The common signaling mechanism that emerges from these studies suggests a model for cortisol secretion similar to that previously proposed for ACTH and AII, wherein I_AC inhibition leads to membrane depolarization, Ca⁺⁺ entry through T-type channels, and cortisol secretion (Enyeart et al., 1993).

Nucleotide Receptor Subtype. Patch clamp experiments showed that the nucleotide receptor whose activation was linked to the inhibition of I_AC K⁺ current and membrane depolarization in AZF cells was a G protein-coupled receptor of the P2Y family. The results clearly demonstrate that ATP and other nucleotides do not function by activation of nonselective cation channels. In many cells, ATP-induced membrane depolarization is mediated through activation of these P2X receptors (Dalziel and Westfall, 1994; Williams and Burnstock, 1997). However, in the course of our studies, ATP and other adenine nucleotides were applied to more than 200 AZF cells at a holding potential of 80 mV. An inward current was never activated in any of these cells upon superfusing the nucleotides.

Modulation of Other K⁺ Channels by Nucleotides. Several reports have appeared demonstrating the modulation of specific K⁺ channels by activation of G protein-coupled purinergic receptors. In cardiac myocytes, several different varieties of K⁺ channels are modulated by purinergic receptor activation (Matsuura and Ehara, 1996; Matsuura et al., 1996). Ca⁺⁺-activated K⁺ channels are activated by purinergic agonists in smooth muscle cells and in rat hepatocytes (Yamashita et al., 1996; Vogalis and Goyal, 1997). In none of these cells was the K⁺ channel modulated by pyrimidine nucleotides. Thus, I_AC K⁺ channels are distinctive in their inhibition through P2Y₃ or closely related receptors.

PLC-Independent Signaling Pathway. Phospholipase C activation is the major signaling pathway that links various P2Y receptors to cellular responses (Dubyak and El-Moatassim, 1993; Filtz et al., 1997). Accordingly, extracellular ATP and UTP stimulate membrane phosphoinositol turnover and release of intracellular Ca⁺⁺ in AZF cells (Niitsu, 1992; Hoey et al., 1994). However, ATP-mediated inhibition of I_AC appears to occur through an alternative signaling pathway. PLC-mediated responses usually are mediated through PKC or Ca⁺⁺, acting either directly or through a calmodulin-dependent process (Berridge, 1993). The complete inability of the potent kinase C antagonist calphostin C to suppress ATP-mediated inhibition of I_AC when applied directly to the cytoplasm through the patch electrode at 10 times the reported IC₅₀ is convincing evidence that activation of this enzyme is not necessary for this response. However, we cannot state with certainty that PKC was inhibited completely in these experiments.

I_AC Inhibition and ATP Hydrolysis. Inhibition of I_AC by external ATP also was prevented by substitution of the nonhydrolyzable ATP analog AMP-PNP or the pyrimidine UTP for ATP in the pipette. This result is not surprising, in view of the fact that neither of these nucleotides acts as a substrate for protein kinases (Azhar and Menon, 1975; Krebs and Beavo, 1979). However, the ineffectiveness of external ATP when the pipette contained 200 µM MgATP in addition to 5 mM UTP was unexpected. Protein kinases are fully activated by ATP at concentrations less than 50 µM (Glynn and Hoffman, 1971; Lemaire et al., 1974), whereas cellular ATPases have K_m values for ATP of several millimolar (Hilgemann, 1997). Thus, inhibition of I_AC by nucleotide receptor activation may require both active kinases as well as ATPases, as appears to be the case for the cystic fibrosis transmembrane conductance regulator Cl channel, where channel gating is fueled by the energy of ATP hydrolysis but depends also on channel phosphorylation by a protein kinase (Baukrowitz et al., 1994). Alternatively, UTP might act as a competitive antagonist of ATP at its binding site on the protein kinase responsible for I_AC inhibition. However, to our knowledge, UTP has not been shown to inhibit protein kinases.

I_AC Inhibition: Comparison of ATP, ACTH, and AII Signaling Pathways. ATP-mediated inhibition of I_AC occurs through a mechanism that is clearly different from that of ACTH. In contrast to ATP, inhibition of I_AC by ACTH is unaffected by staurosporine. Furthermore, when I_AC is activated by pipette solutions containing 5 mM UTP, 50 µM MgATP in the pipette is sufficient to restore complete inhibition of I_AC by ACTH (100 pM). Under the same conditions, externally applied ATP (10 µM) produced no inhibition of I_AC.

Nucleotide-Mediated Inhibition of I_AC and Membrane Depolarization. Bovine AZF cells express three types of ion channels, including rapidly inactivating Ca⁺⁺ and K⁺ channels and noninactivating K⁺ channels (Mlinar et al., 1993a,b; Mlinar and Enyeart, 1993). Of these, only I_AC channels are open at negative membrane potentials and exhibit little voltage-dependent gating. It is likely that these channels are largely responsible for determining the resting potential of these cells (Mlinar et al., 1993a; Enyeart et al., 1996; Enyeart et al., 1997). Accordingly, ACTH, AII, ATP, and UTP all depolarize AZF cells at concentrations that maximally inhibit I_AC. Thus, nucleotides and peptide hormones that inhibit I_AC through activation of three different receptors share the ability to depolarize AZF cells and stimulate large (>50-fold) increases in cortisol secretion (Enyeart et al., 1993; Mlinar et al., 1993a; Mlinar et al., 1995).

Physiological Relevance. The physiological significance of external ATP or other nucleotides in the regulation of cortisol secretion is not well established. ATP and other purines are stored and released, along with catecholamines, from secretory granules of adrenal medullary chromaffin cells (Glynn and Hoffman, 1971). In addition to nucleotide receptors, adrenal zona fasciculata cells also express -adrenergic receptors that, when activated, stimulate cortisol secretion (Kawamura et al., 1984; Walker et al., 1988). Rays of adrenal medullary tissue have been reported to traverse the adrenal cortex, and clusters of chromaffin cells have been reported to exist in all three regions of the adrenal cortex (Nussdorfer, 1996). Overall, these results suggest that ATP and other nucleotides released from adrenal chromaffin cells, along with catecholamines, may act in a paracrine fashion to modulate I_AC current and cortisol secretion in bovine AZF cells.