Department of Neuroscience, The Ohio State University, College of Medicine, Columbus, Ohio 43210
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ABSTRACT |
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Bovine adrenal zona fasciculata cells express background K+ channels (IAC channels) whose activity is potently inhibited by ACTH. In whole cell patch clamp recordings, it was discovered that the trivalent lanthanides (Ln3+s) lanthanum and ytterbium interact with two binding sites to modulate K+ flow through these channels. Despite large differences in ionic radii, these Ln3+s inhibited IAC channels half-maximally with IC50 values near 50 µM. In addition, these Ln3+s blocked and reversed ACTH-mediated inhibition of IAC K+ channels at similar concentrations. The Ln3+s did not alter inhibition of IAC by angiotensin II or cAMP. Ln3+-induced uncoupling of ACTH receptor activation from IAC inhibition was prevented by raising the external Ca2+ concentration from 2 to 10 mM. The divalent cation Ni2+ (500 µM) also blocked ACTH-dependent inhibition of IAC through a Ca2+-sensitive mechanism. The results are consistent with a model in which Ln3+s produce opposing actions on IAC K+ currents through two separate binding sites. In addition to directly inhibiting IAC, Ln3+s (and Ni2+) bind with high affinity to a Ca2+-selective site associated with the ACTH receptor. By displacing Ca2+ from this site, Ln3+s prevent ACTH from binding and accelerate its dissociation. These results identify Ln3+s as a relatively potent group of noncompetitive ACTH receptor antagonists. Allosteric actions of trivalent and divalent metal cations on hormone binding, mediated through Ca2+-specific sites, may be common to a variety of peptide hormone receptors.
K+ channels; nickel; calcium; adrenocorticotropic hormone receptor
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INTRODUCTION |
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THE LANTHANIDES (Ln3+s; elements 58-71 in the periodic table) are trivalent metal cations that interact with a range of biologically important proteins, including ion channels and G protein-coupled receptors. In many cases, the strong interaction of Ln3+s with these proteins occurs because these agents share biologically important properties with the divalent calcium (Ca2+) cation (12, 27, 38). Their similarity to Ca2+ with respect to ionic radii, coordination chemistry, and affinity for the oxygen donor groups underlies their strong interaction with Ca2+-binding sites on a wide range of proteins (7, 12, 27).
Ln3+s have been particularly valuable in the study of voltage-gated ion channels, including Ca2+ and K+ channels. Ln3+s inhibit low-voltage-activated T-type Ca2+ channels by pore occlusion with a potency that varies inversely with ionic radius (24). In contrast, Ln3+s inhibit L-type Ca2+ channels with a potency that varies directly with ionic radius (19). For both Ca2+ channel subtypes, Ln3+ effects are produced through interaction with Ca2+-binding sites.
Ln3+s also suppress ion flow through voltage-gated
K+ channels with a potency that varies inversely with the
ionic radius (11). However, the suppression of
K+ currents by Ln3+s appears to occur by an
entirely different mechanism. Rather than blocking K+
channels by pore occlusion, Ln3+s suppress K+
currents by altering voltage-dependent gating and kinetic parameters by
interaction with binding sites that are not Ca2+ specific
(11, 36). An additional effect on K+
permeation has not been ruled out. In this regard, several
non-voltage-gated background K+ channels are inhibited by
the Ln3+ gadolinium (Gd3+) with
IC50 values 100 µM (20, 29).
However, an inward rectifier in rat corticotropes is insensitive to
La3+ (18).
In addition to ion channels, Ca2+-specific and nonspecific Ln3+-binding sites have been identified on several membrane receptors. Acetylcholine and insulin receptors possess two types of Ln3+-binding sites, only one of which accepts Ca2+ (30, 38). Ln3+s bind with high affinity to extracellular Ca2+-sensing receptors on bovine parathyroid cells (2).
Bovine adrenal cortical cells, including those of the fasciculata and glomerulosa, express ACTH receptors whose activation is coupled to the inhibition of a unique population of ATP-dependent background K+ channel (IAC) (8, 22). These IAC K+ channels set the resting potential of adrenal zona fasciculata (AZF) cells and couple ACTH receptors to depolarization-dependent Ca2+ entry and cortisol secretion (9, 22). A requirement for Ca2+ in the binding of ACTH to its receptor on these cells, as well as its continued receptor occupancy, has been well established (6). Optimum binding requires the presence of millimolar Ca2+. Accordingly, in the absence of extracellular Ca2+, ACTH fails to inhibit IAC K+ channels (10). The mechanism that underlies the obligatory requirement for Ca2+ in ACTH binding and receptor occupation has not been determined.
In addition to trivalent cations, divalent cations including Ni2+ also interact with Ca2+-binding sites on membrane channel proteins. Ni2+ preferentially blocks low-voltage-activated T-type Ca2+ channels (15, 24). Ni2+ also modulates ion flux through voltage-gated K+ channels and cyclic nucleotide-gated channels (13, 25).
Using whole cell patch clamp recording, we have studied the interaction of Ln3+s of large (La3+) and small (Yb3+) ionic radii and Ni2+ on K+ current through IAC channels in the absence and in the presence of ACTH.
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MATERIALS AND METHODS |
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Tissue culture media, antibiotics, fibronectin, and fetal calf
serum (FCS) were obtained from GIBCO-BRL (Grand Island, NY). Culture
dishes were purchased from Corning (Corning, NY). Coverslips were from
Bellco (Vineland, NY). Lanthanide chlorides (99.9% purity) were
obtained from Aldrich Chemical (Milwaukee, WI). All other chemicals
were purchased from Sigma (St. Louis, MO).
Isolation and culture of AZF cells. Bovine adrenal glands were obtained from steers (age range 1-3 yr) within 30 min of slaughter at a local slaughterhouse. Fatty tissue was removed immediately, and the glands were transported to the laboratory in ice-cold PBS containing 0.2% dextrose. Isolated AZF cells were prepared as previously described (10). After isolation, cells were either resuspended in DMEM-F12 (1:1) with 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and the antioxidants 1 µM tocopherol, 20 nM selenite, and 100 µM ascorbic acid (DMEM-F12+) and plated for immediate use or resuspended in FCS-5% DMSO, divided into 1-ml aliquots each containing ~2 × 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 cells were added. Dishes were maintained in DMEM-F12+ at 37°C in a humidified atmosphere of 95% air-5% CO2.
Solutions and bath perfusion.
For recording whole cell K+ currents, the standard pipette
solution was 120 mM KCl, 2 mM MgCl2, 1 mM
CaCl2, 10 mM HEPES, 11 mM
1,2-bis-(2-aminophenoxy)ethane-N,N,N',N"-tetraacetic acid, 200 µM GTP, and 5 mM MgATP, with pH buffered to 7.2 using KOH. With
this composition, the free Ca2+ concentration was
determined to be 2.3 × 108 M by use of the "Bound
and Determined" program (1). Pipette solutions were
filtered through 0.22-µm cellulose acetate filters. The external
solution consisted of (in mM) 140 NaCl, 5 KCl, 2 CaCl, 2 MgCl2, 10 HEPES, and 5 glucose, pH 7.35, using NaOH.
Recording conditions and electronics.
AZF cells were used for patch clamp experiments 2-12 h after
plating. Coverslips with cells were transferred from 35-mm culture dishes to the recording chamber. Cells with diameters of 10-15 µm and capacitances of 8-15 pF were used for
recording. Patch electrodes with resistances of 1-2 M were
fabricated from 0010 glass (Corning) using a Brown-Flaming model P-87
microelectrode puller (Sutter Instruments, Novato, CA). Access
resistance during recording, estimated from the transient cancellation
controls of the patch clamp amplifier, was 2-5 M
. The
combination of access resistance and cell capacitance yielded voltage
clamp time constants of <100 µs.
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RESULTS |
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Bovine AZF cells express two types of K+ current. These include a voltage-gated, rapidly inactivating Kv1.4 current and a noninactivating background K+ current that is activated by intracellular ATP (8, 22, 25). In whole cell recordings, IAC grows dramatically over a period of minutes, provided that ATP or other nucleotides are present at millimolar concentrations in the recording pipette (8, 22).
The absence of time- and voltage-dependent inactivation of
IAC K+ channels allows the
corresponding membrane current to be easily isolated in whole cell
recording with the use of either of two voltage clamp protocols. When
voltage steps of 300 ms duration are applied from a holding potential
of 80 mV, IAC can be measured near the end of
a voltage step when the transient Kv1.4 current has inactivated (Fig.
1A, left traces).
Alternatively, IAC can be selectively activated
by an identical voltage step after a 10-s prepulse to
20 mV has fully
inactivated the Kv1.4 current (Fig. 1A, right
traces). Measurement of IAC by either method
yielded nearly identical results.
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Inhibition of IAC by Yb3+ and La3+. To determine whether Ln3+s inhibited K+ flow through the non-voltage-gated background IAC K+ channels, we examined the effects of the largest Ln3+s, La3+ (ionic radius 1.160 Å), and one of the smallest Ln3+s, Yb3+ (ionic radius 0.985 Å), on whole cell IAC currents in AZF cells. Both of these Ln3+s produced concentration-dependent inhibition of IAC K+ current (Fig. 1). In contrast to their effect on the voltage-gated Kv1.4 current (11), these two Ln3+s inhibited IAC at nearly identical concentrations, with IC50s for La3+ and Yb3+ of 52.5 and 50.1 µM, respectively (Fig. 1C).
Ln3+-induced increase in IAC. Although La3+ and Yb3+ inhibited IAC with similar potency, it was observed that the initial inhibition of IAC produced by these two agents was often followed by a delayed increase in IAC amplitude (Fig. 1B in 10 µM Yb3+). The increase in IAC occurred even though this current had clearly reached a stable maximum value previous to Ln3+ exposure.
To further explore the biphasic effect of Ln3+s on IAC current amplitude, IAC was allowed to reach a stable maximum value before the AZF cells were superfused with Ln3+ (50 µM) for 10 min. Ln3+ was then washed from the chamber with control saline (Fig. 2). Under these conditions, Yb3+ and La3+ produced similar effects. The initial rapid inhibition (Fig. 2, A and B, trace 3) was followed by a gradual increase in current amplitude that persisted for a period of minutes (Fig. 2, A and B, trace 4). Subsequent superfusion of control saline immediately reversed inhibition, and IAC rapidly increased to a value significantly greater than that reached before Ln3+ application (Fig. 2, trace 5). In the two experiments illustrated in Fig. 2, IAC increased by 66 and 63%, respectively. Overall, a 10-min exposure to 50 µM Yb3+ or La3+ followed by washing increased IAC amplitude significantly in 6 of 11 and 5 of 7 cells, respectively.
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Ln3+s reverse ACTH-mediated inhibition of IAC. The peptide hormone ACTH inhibits IAC half-maximally at a concentration of 5.4 pM (22). The requirement for Ca2+ in ACTH binding and inhibition (6, 10), combined with the high affinity of Ln3+s for some Ca2+-binding sites, raised the possibility that Ln3+-stimulated enhancement of IAC current occurred through interaction of the Ln3+ with a Ca2+-binding site located on the ACTH receptor.
To test this possibility, we studied the effects of Ln3+s on reversal of ACTH-mediated inhibition of IAC. In the experiment illustrated in Fig. 3A, IAC increased to a stable maximum value after ~20 min of whole cell recording (trace 2). Superfusion of 200 pM ACTH produced near complete inhibition of IAC (trace 3) by a mechanism that was not measurably reversed by a 10-min exposure to control saline. Subsequent superfusion with saline containing 50 µM LaCl3 produced a slow reversal of the ACTH-mediated inhibition (trace 4), an effect that was partially masked in time and extent by the separate inhibitory action of La3+ on IAC. A second wash with control saline reversed the La3+ inhibition, at which time IAC was restored to 84% of its original maximum value (trace 5).
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Suppression of ACTH-mediated IAC inhibition by
Ln3+s and
Ni2+.
In addition to reversing the ACTH-mediated inhibition of
IAC, pretreatment of cells with
Ln3+s produced a concentration-dependent suppression of
IAC inhibition by ACTH. In the
experiment illustrated in Fig.
4A, cells were superfused with
either 10 (left) or 50 µM (right)
La3+ before cells were superfused with La3+ and
ACTH (200 pM) in combination. At a concentration of 10 µM, La3+ failed to alter the complete inhibition of
IAC by ACTH (left). However, at a
concentration of 50 µM, La3+ completely prevented
IAC inhibition by ACTH (right).
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Antagonism of ACTH inhibition of IAC by
Ni2+.
The divalent cation Ni2+ blocks T-type Ca2+
channels and voltage-gated Kv1.4 K+ channels in AZF cells
with respective IC50 values of 5.7 and 467 µM (24,
25). By comparison, at concentrations 500 µM, Ni2+ was ineffective as an inhibitor of
IAC K+ current. However, 500 µM
Ni2+ nearly eliminated IAC
inhibition by ACTH (200 pM).
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La3+ and Ni2+ do not alter IAC inhibition by ANG II. In addition to ACTH, IAC K+ channels are potently inhibited by the peptide hormone ANG II with an IC50 of 150 pM (22, 23). Experiments with ANG II showed that the block of ACTH-mediated IAC inhibition by La3+ and Ni2+ was specific and not a generalized effect on peptide hormone receptors.
In the experiment illustrated in Fig. 6A (left), IAC was allowed to reach a stable amplitude before the cell was sequentially superfused with saline containing La3+ (50 µM) alone followed by La3+ plus ACTH (200 pM) and, finally, La3+ plus ANG II (10 nM). In the presence of 50 µM La3+, ACTH was completely ineffective at inhibiting IAC, whereas ANG II inhibited IAC almost completely.
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La3+ and Ni2+ do not affect inhibition of IAC by cAMP. La3+ and Ni2+ could potentially act at several different locations in the signaling pathway to block ACTH-mediated inhibition of IAC. If the site of action of the metals was proximal to the activation of adenylate cyclase, inhibition of IAC by cAMP should not be affected. Accordingly, it was discovered that La3+ and Ni2+ were ineffective at preventing inhibition of IAC by the membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (8-pcpt-cAMP).
In the experiment illustrated in Fig. 7A, the cell was preexposed to saline containing La3+ before sequential superfusion of saline containing La3+ (50 µM) and ACTH (200 pM) or 8-pcpt-cAMP (500 µM). In this experiment, La3+ reduced IAC amplitude by 47%, whereas ACTH failed to produce any further inhibition in the presence of La3+. The subsequent superfusion of 8-pcpt-cAMP inhibited IAC completely. As shown in Fig. 7B, 8-pcpt-cAMP inhibited IAC by 84 ± 2% (n = 3) in the presence of 50 µM La3+ compared with 74 ± 7% (n = 3) in control saline.
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Ca2+ antagonizes effects of La3+ and Ni2+. The continued effectiveness of 8-pcpt-cAMP in inhibiting IAC in the presence of La3+ indicated that this cation blocks ACTH action at a step that precedes the activation of adenylate cyclase. In view of the requirement for Ca2+ in ACTH binding and the previously mentioned requirement for Ca2+ in ACTH-mediated inhibition of IAC, it is possible that La3+ could antagonize the binding of this peptide by occupying a specific Ca2+-binding site associated with the ACTH receptor. If so, then raising extracellular Ca2+ should competitively antagonize the La3+ block of IAC inhibition by ACTH.
When extracellular Ca2+ was raised from 2 to 10 mM, La3+ (50 µM) was completely ineffective at suppressing ACTH-mediated inhibition of IAC. In the experiment illustrated in Fig. 8A, the cell was superfused with control saline containing 10 mM Ca2+. In this high-Ca2+-containing external solution, La3+ failed to blunt the complete inhibition of IAC by 200 pM ACTH. Overall, in saline containing 10 mM Ca2+ and 50 µM La3+, ACTH (200 pM) inhibited IAC by 96 ± 4% (n = 3), a value not significantly different from that produced by ACTH alone in standard (2 mM Ca2+) saline. By comparison, in standard saline containing 50 µM La3+, ACTH was far less effective, inhibiting IAC by only 2 ± 1% (n = 9) (Fig. 4A).
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DISCUSSION |
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In this study, it was discovered that Ln3+s produce dual effects on K+ current through IAC background K+ channels in AZF cells. In addition to directly inhibiting IAC, the Ln3+s block and reverse ACTH-mediated inhibition of these channels. These opposing actions are mediated through distinct binding sites associated with IAC K+ channels and ACTH receptors. Most interestingly, Ln3+s appear to uncouple ACTH receptors from IAC K+ channel inhibition by competitively antagonizing the binding of Ca2+ to a specific site. By displacing Ca2+ from this site, which is required for ACTH binding or receptor activation, Ln3+s act as potent noncompetitive ACTH antagonists. The divalent cation Ni2+ antagonized the actions of ACTH in a similar fashion but was ~10-fold less potent than the Ln3+s.
Block of IAC K+ channels by Ln3+s. The results in this study show that Ln3+s inhibit IAC K+ channels through a mechanism that differs in at least two fundamental respects from their inhibition of voltage-gated bKv1.4 K+ channels. Specifically, Ln3+s reduced bKv1.4 K+ current by altering voltage-dependent gating and kinetic parameters (11). Because IAC channels exhibit little or no voltage dependence, the inhibition of these channels by La3+ and Yb3+ must have occurred through a different mechanism, probably by pore occlusion.
Second, with regard to potency, Yb3+ and La3+ inhibited IAC K+ channels with almost identical potency, despite the marked difference in ionic radii (33). By comparison, Ln3+s inhibit bKv1.4 K+ channels with a potency that varies inversely with ionic radius (11). Because both of these Ln3+s at low concentrations inhibited bKv1.4 current by actions on gating, we were unable to determine whether these agents also inhibit K+ permeation through voltage-gated channels. In this regard, voltage-gated K+ channels and background K+ channels have distinct pore structures and exhibit different pharmacology (20, 29) The molecular identity of the IAC channel is unknown, but it displays several properties of background K+ channels, which are structurally characterized by four membrane-spanning regions and two pore domains. In this regard, several members of this family of background K+ channels are blocked by the Ln3+ Gd3+ with potency similar to what we observed for the block of IAC by Ln3+s (20, 29).Ln3+ inhibition of ACTH action. The studies demonstrating the reversal and block of ACTH-mediated inhibition of IAC K+ channels by Ln3+s suggest a model in which these cations displace Ca2+ from a specific site on the ACTH receptor, allosterically producing a marked reduction in the affinity of the receptor for ACTH. The results of our study are consistent with this model, given well established quantitative measurements relating ACTH receptor activation to IAC inhibition and assuming only that the potent inhibitory action of ACTH on IAC channels requires the continued occupancy of the receptor by this peptide.
Within this framework, superfusing AZF cells with saline containing Ln3+s rapidly reversed ACTH-mediated inhibition of IAC, when normal saline did not, because replacement of Ca2+ with Ln3+ lowered the affinity of the receptor for ACTH, leading to the peptide's rapid dissociation. Similarly, ACTH was ineffective at inhibiting IAC in experiments where cells were preexposed to Ln3+s, because Ln3+-bound receptors have a low affinity for ACTH. This model also explains the novel biphasic effect of Ln3+ in which rapid IAC inhibition was often followed by a slower increase in IAC amplitude. Presumably, the serum-supplemented culture medium contained sufficient ACTH to partially inhibit IAC current. This inhibition was unmasked only when the residual ACTH dissociated in the presence of Ln3+s. The FCS used in our culture medium is not assayed for ACTH. However, the presence of only 1 × 10Quantitative aspects of effects of
Ln3+s on ACTH binding.
Results from previous studies indicated that ACTH must occupy only a
small fraction of ACTH receptors to completely inhibit IAC K+ current (3, 5,
22). Specifically, ACTH binds to a single type of ACTH receptor
on AZF cells with a dissociation constant (Kd)
of ~1.5 nM (3, 5), whereas IAC is
inhibited half-maximally by ACTH at 5.4 pM (22).
Substitution of these values into an equation of the Langmuir
absorption isotherm form indicates that activation of only 0.4% of
ACTH receptors would result in 50% inhibition of
IAC. Thus the observed effectiveness of 50 µM
La3+ or Yb3+ in completely blocking ACTH
inhibition of IAC indicates that, at this
concentration, 99.5% of ACTH receptors are occupied by Ln3+ and cannot be activated by ACTH.
Molecular mechanism of Ln3+ antagonism of ACTH action. The results of our study are consistent with the hypothesis that Ln3+s competitively antagonize the binding of Ca2+ to a specific site located on the ACTH receptor. The displacement of Ca2+ from this site dramatically lowers the affinity of the receptor for ACTH, preventing its binding and accelerating ACTH dissociation. Because of their high-affinity binding to this Ca2+ site, Ln3+s act as potent noncompetitive antagonists of ACTH in AZF cells.
Our findings are consistent with and extend those of a previous study that demonstrated an obligatory role for Ca2+ in ACTH binding and steroidogenesis. Specifically, Cheitlin et al. (6) showed that Ca2+ at millimolar concentrations was required for optimum ACTH binding to rat AZF receptors and that removal of all external Ca2+ markedly accelerated ACTH dissociation from its receptor, reducing the t1/2 from 32 to 3.5 min. Although this study demonstrated a requirement for Ca2+ in ACTH binding, it did not provide evidence for a specific Ca2+-binding site that is linked to the ACTH receptor. For example, according to surface potential theory, Ca2+ at millimolar concentrations could influence ACTH binding by screening negative surface charges on the membrane and electrostatically influencing the interaction of ACTH with its receptor. The present study provides evidence that Ln3+s interact with a specific binding site associated with the ACTH receptor. The observation that the addition of Ln3+s at micromolar concentrations is as effective as Ca2+ removal in blocking ACTH inhibition of IAC suggests a common mechanism involving displacement of Ca2+ from a specific site. With regard to the specificity of the Ln3+-binding site, our experimental design did not detect a difference in potency between La3+ and Yb3+, which span nearly the entire range of ionic radii for these elements (33). These results are in contrast to the large size-dependent differences in potency observed for Ln3+ inhibition of ion channels and indicate significant differences in these binding sites (11, 24).Comparison with Ln3+ effects in other biological systems. The actions of Ln3+s in AZF cells parallel their effects in other biological systems. Despite their high affinity for Ca2+-binding sites, the Ln3+s have no known biological function. Indeed, when Ln3+s interact with Ca2+-specific and nonspecific sites on proteins, ranging from enzymes to receptors to ion channels, their action is generally inhibitory (12, 19, 24). In one exceptional example, terbium (Tb3+) has been shown to enhance insulin binding to its receptor by high-affinity interaction with a Ca2+-binding site (37).
Although it appears likely that Ln3+s antagonize ACTH binding to its receptor through interaction with a Ca2+-binding site, other possibilities cannot be ruled out. For example, it is possible that Ln3+s do not inhibit ACTH binding to its receptor, but instead interfere with subsequent steps required for the production of cAMP. Furthermore, a direct interaction of Ln3+s with ACTH seems unlikely but cannot be excluded. The number of Ca2+-dependent G protein-coupled receptors whose activity could be modulated by Ln3+s as well as divalent cations is unknown. ACTH receptors belong to the melanocortin receptor subfamily that includes multiple receptors for melanocyte-stimulating hormone (26). It is possible that Ln3+s and Ni2+ would also inhibit hormone binding to all of these receptors through interaction with Ca2+-binding sites.Effect of Ni2+ on ACTH response. Even though Ni2+ failed to inhibit IAC K+ channels, it suppressed ACTH inhibition of IAC, although it was 10-fold less potent than Ln3+s in this respect. Thus the effects of Ni2+ on IAC were mediated through a single site associated with the ACTH receptor. The inhibitory effects of Ni2+ on voltage-gated K+ channels occur through actions on gating rather than permeation (11, 35). Apparently, these binding sites are missing on non-voltage-gated background channels such as IAC.
It is likely that Ni2+ and Ln3+s inhibit ACTH receptor activation through interaction with a common binding site. Ni2+ and Ln3+s inhibit a number of ion channels through Ca2+-binding sites. Invariably, the Ln3+s are 10- to 100-fold more potent in this respect (11, 19, 24).Use of Ln3+s and Ni2+ as Ca2+ antagonists. Ln3+s and Ni2+ and other divalent cations have been used as Ca2+ antagonists in a range of studies to explore the role of voltage-gated Ca2+ entry in peptide hormone-stimulated secretion (4, 17). These include studies of ACTH-stimulated cortisol secretion (9, 21, 31, 32, 34). Results of our study indicate that interpretation of these experiments is complicated by the inhibitory actions of these metal cations on ACTH receptors. Finally, they also suggest that Ln3+s as well as divalent cations may produce toxic actions on the adrenal gland through inhibitory actions on ACTH binding. Toxic effects of metals on adrenal steroidogenesis have been reported (28).
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant no. DK-47875 to J. J. Enyeart.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. J. Enyeart, Dept. of Neuroscience, The Ohio State University, College of Medicine, 5190 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239 (E-mail: enyeart.1{at}osu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 26, 2002;10.1152/ajpendo.00478.2001
Received 25 October 2001; accepted in final form 18 February 2002.
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