Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Juan Carlos Gomora1 and John J. Enyeart1,2

1 Department of Pharmacology and 2 Neuroscience Program, Ohio State University College of Medicine, Columbus, Ohio 43210-1239

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Bovine adrenal zona fasciculata cells (AZF) express a noninactivating K+ current (IAC) whose inhibition by adrenocorticotropic hormone and ANG II may be coupled to membrane depolarization and Ca2+-dependent cortisol secretion. We studied IAC inhibition by Ca2+ and the Ca2+ ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette) Ca2+ concentration ([Ca2+]i) buffered to 0.02 µM, IAC reached maximum current density of 25.0 ± 5.1 pA/pF (n = 16); raising [Ca2+]i to 2.0 µM reduced it 76%. In inside-out patches, elevated [Ca2+]i dramatically reduced IAC channel activity. Ionomycin inhibited IAC by 88 ± 4% (n = 14) without altering rapidly inactivating A-type K+ current. Inhibition of IAC by ionomycin was unaltered by adding calmodulin inhibitory peptide to the pipette or replacing ATP with its nonhydrolyzable analog 5'-adenylylimidodiphosphate. IAC inhibition by ionomycin was associated with membrane depolarization. When [Ca2+]i was buffered to 0.02 µM with 2 and 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibited IAC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibited IAC significantly more effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising [Ca2+]i inhibits IAC through a mechanism not requiring calmodulin or protein kinases, suggesting direct interaction with IAC channels. ANG II may inhibit IAC and depolarize AZF by activating parallel signaling pathways, one of which uses Ca2+ as a mediator.

adrenal cortex; potassium channel; angiotensin II; membrane depolarization; cortisol secretion

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

BOVINE ADRENAL ZONA FASCICULATA cells (AZF cells) express a noninactivating K+ current (IAC) that appears to set the resting potential of these cells and to function critically in the regulation of cortisol secretion (20). These IAC channels show little voltage dependence and are open at negative membrane potentials (6, 8, 20). IAC channel activity is regulated by diverse hormonal and metabolic factors. Specifically, these channels are activated by hydrolyzable and nonhydrolyzable forms of ATP over a physiological range of concentrations, suggesting that IAC channels may function as metabolic sensors linking the metabolic state of the cell to membrane potential (6).

IAC is inhibited by adrenocorticotropic hormone (ACTH) and ANG II at concentrations identical to those that trigger membrane depolarization and cortisol secretion (7, 20). ACTH and ANG II each depolarize AZF cells by a maximum of >50 mV (20). The signaling pathways that link ACTH and ANG II receptors to IAC inhibition have been partially characterized. ACTH inhibits IAC completely by a cAMP-dependent mechanism that appears to be independent of increases in intracellular Ca2+ concentration ([Ca2+]i) (8, 20). ANG II inhibits IAC through activation of a losartan-sensitive AT1 receptor that is coupled to activation of phospholipase C (PLC) and the release of Ca2+. ANG II produces only partial inhibition (~75%) of IAC under conditions in which [Ca2+]i is strongly buffered by 11 or 20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (20, 22). Although this partial inhibition of IAC by ANG II may be independent of Ca2+, a Ca2+-dependent mechanism may be necessary for complete inhibition leading to membrane depolarization.

Ca2+ modulates the activity of a number of different K+ channels. Several types of Ca2+-activated K+ channels are expressed by a variety of excitable and nonexcitable cells (12). Recently, K+ channels whose activity is inhibited by Ca2+ have been identified in cells, including neurons and lymphocytes (1, 29, 31). The modulation of K+ channel activity by Ca2+ may occur through one of many different mechanisms. These include allosteric modulation of K+ channel gating through a direct interaction of Ca2+ with the channels, as occurs with both Ca2+-activated and Ca2+-inhibited K+ channels (5, 27, 28). Alternatively, Ca2+ might function through activation of any of several Ca2+-dependent enzymes, including protein kinase C (PKC), or Ca2+/calmodulin-activated enzymes, including calmodulin kinase II and the Ca2+-activated phosphatase calcineurin (16, 18). Direct modulation of cyclic nucleotide-gated, nonselective cation channels by Ca2+/calmodulin has also been reported (17).

We have studied the inhibition of IAC by Ca2+ in whole cell patch-clamp recordings from bovine AZF cells. Ca2+ inhibits IAC channels by a mechanism that is independent of calmodulin and protein kinases but is tightly linked to membrane depolarization.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Tissue culture media, antibiotics, fibronectin, and fetal bovine serum (FBS) were obtained from GIBCO Laboratories (Grand Island, NY). Coverslips were from Bellco Glass (Vineland, NJ). Enzymes, ACTH (1---24), ANG II, MgATP, 5'-adenylylimidodiphosphate (AMP-PNP; lithium salt), NaGTP, BAPTA, and ionomycin were obtained from Sigma Chemical (St. Louis, MO). Calmodulin inhibitory peptide (residues 290-309 of calmodulin kinase II) was obtained from Biomol (Plymouth Meeting, PA).

Isolation and culture of AZF cells. Bovine adrenal glands were obtained from steers (age range 1-3 yr) within 15 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 (9), 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 large pieces of ~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 or two subsequent fasciculata slices were saved in cold sterile PBS-0.2% dextrose. The fasciculata/glomerulosa margin (~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 (~200-300 U/ml) type I collagenase (neutral protease activity not exceeding 100 U/mg of solid) and 0.2 mg/ml deoxyribonuclease in DMEM-F12 for ~1 h at 37°C, with trituration 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 100 g for 5 min. Undigested tissue remaining in the cheesecloth was collagenase treated for an additional 1 h. Pelleted cells were washed twice with DMEM-0.2% BSA and centrifuged 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% 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 ~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 addition of 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 and inside-out patch configurations. For whole cell recordings, the standard external solution consisted of (in mM) 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 5 glucose, with pH buffered to 7.4 using NaOH. The standard pipette solution was (in mM) 120 KCl, 2 MgCl2, 10 HEPES, and 5 MgATP, as well as 200 µM GTP, with pH buffered to 7.2 using KOH. The buffering capacity and Ca2+ concentration of the pipette solutions was varied by adding combinations of BAPTA and CaCl2 using the Bound and Determined program (2). Low- and high-capacity Ca2+-buffering solutions contained 2 and 11 mM BAPTA, respectively. In most experiments, [Ca2+]i was buffered to 0.02 µM. Variations are noted in the text. All solutions were filtered through 0.22-µm cellulose acetate filters. For inside-out patch recordings, the standard external and pipette solutions used in whole cell recordings were switched.

AZF cells were used for patch-clamp experiments 2-12 h after plating. Typically, cells with diameters of 10-15 µm and capacitances of 8-15 pF were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume 1.5 ml), which was continuously perfused by gravity at a rate of 3-5 ml/min. For whole cell recordings, patch electrodes with resistances of 1.0-2.0 MOmega were fabricated from Corning 0010 glass (Garner Glass, Claremont, CA). These routinely yielded access resistances of 1.5-4 MOmega and voltage-clamp time constants of <100 µs. For single-channel recordings, patch electrodes with higher resistances of 3-5 MOmega were used. K+ currents were recorded at room temperature (22-25°C), following the procedure of Hamill et al. (10) and using a List EPC-7 patch-clamp amplifier.

Pulse generation and data acquisition were done using a personal computer and pCLAMP software with a TL-1 interface (Axon Instruments, Burlingame, CA). Currents were digitized at 1-20 kHz after filtering with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using scaled hyperpolarizing steps of one-half to one-fourth amplitude. Data were analyzed and plotted using pCLAMP 5.5 and 6.02 (Clampan, Clampfit, Fetchan, and Pstat), SigmaPlot 3.0, and GraphPad InPlot 4.03. Drugs were applied by bath perfusion that was controlled manually by a six-way rotary valve.

Series resistance compensation was not used in most experiments. The mean amplitude of IAC in AZF cells was <500 pA. A current of this size in combination with a 4-MOmega access resistance produces a voltage error of only 2 mV, which was not corrected.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

[Ca2+]i and IAC expression. As previously reported, bovine AZF cells express two types of K+ currents: a rapidly inactivating A-type K+ current (IA) (23) and a noninactivating K+ current (IAC) whose amplitude increases continuously over many minutes in whole cell recordings (8, 20). The expression of IAC requires the presence of ATP at millimolar concentrations in the recording pipette (6). The absence of time-dependent inactivation allows the IAC to be easily isolated for measurement in whole cell recordings, using either of two voltage-clamp protocols. When voltage steps 300 ms in duration were applied from a holding potential of -80 mV to a test potential of +20 mV or +30 mV, IAC could be selectively measured near the end of a step, at a point at which the IA had inactivated entirely (Fig. 1A, left). Using the second protocol, IAC was selectively activated with an identical voltage step after a 10-s prepulse to -20 mV had fully inactivated the IA (Fig. 1A, middle).


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Fig. 1.   Effect of intracellular Ca2+ concentration ([Ca2+]i) on noninactivating K+ current (IAC) expression. K+ currents were activated at 30-s intervals by voltage steps to +20 mV from a holding potential of -80 mV with or without a 10-s prepulse to -20 mV that inactivated A-type K+ current. A: K+ currents were recorded with patch pipettes containing standard solution (see METHODS) with Ca2+ buffered to 0.02 µM (top) or 2 µM [Ca2+]i (bottom) using 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), with or without inactivating prepulses, as indicated by voltage protocol diagrams. Left and middle: traces show currents recorded at indicated times after initiation of whole cell recording. Right: IAC current amplitudes (measured near end of voltage step) were plotted against time; open circle , with depolarizing prepulse; bullet , without prepulse. B: IAC current density (expressed as pA/pF) was obtained by dividing maximum IAC current amplitude obtained in experiments such as those illustrated in A by cell capacitance determined from transient cancellation controls of patch-clamp amplifier. Results are means ± SE for indicated no. of determinations at [Ca2+]i of 0.02, 2, and 10 µM.

The time-dependent increase in IAC amplitude observed in whole cell recordings was markedly inhibited when [Ca2+]i was raised from 0.02 to 2.0 µM with Ca2+ buffered by 11 mM BAPTA (Fig. 1, A and B). Overall, with 0.02 µM [Ca2+]i, after 10-20 min, IAC reached a maximum current density of 25.0 ± 5.1 pA/pF (n = 16). By comparison, with 2 µM [Ca2+]i, IAC reached a maximum density of 5.9 ± 3.0 pA/pF (n = 11), a reduction of ~76%. Increasing [Ca2+]i from 2.0 to 10 µM did not further suppress IAC expression, which grew to a maximum of 6.4 ± 2.0 pA/pF (n = 13; Fig. 1B).

Inhibition of IAC by ionomycin. The time-dependent expression of IAC typically observed in whole cell recordings from AZF cells was suppressed by increasing [Ca2+]i to 2 and 10 µM. To determine whether IAC, once expressed, could be inhibited by Ca2+ influx across the plasma membrane, cells were superfused with the Ca2+ ionophore ionomycin after IAC had reached a stable maximum amplitude.

In this series of experiments, ionomycin was superfused at a concentration of 10 µM, and [Ca2+]i was buffered to 0.02 µM with 2 mM BAPTA. Reducing BAPTA from 11 to 2 mM suppressed maximum IAC expression by ~60%, although [Ca2+]i was maintained at 0.02 µM in each case. With 2 mM BAPTA in the pipette, ionomycin was less effective at concentrations <10 µM. The combination of 2 mM BAPTA and 10 µM ionomycin proved to be best for this study.

As illustrated in Fig. 2, ionomycin (10 µM) selectively inhibited the IAC. Inhibition was detectable after a delay of 2-3 min and progressed to near completion after 10 min (Fig. 2, A and B). In contrast to IAC, the IA current remained after IAC was nearly completely inhibited by ionomycin (Fig. 2A). Overall, ionomycin (10 µM) inhibited IAC by 88 ± 4% (n = 14). Ionomycin-mediated inhibition of IAC was only partially reversible. The current was restored to 44 ± 11% (n = 8) of its maximum value with prolonged washing.


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Fig. 2.   Effect of ionomycin on IAC. K+ currents were activated at 30-s intervals by voltage steps to +30 mV from a holding potential of -80 mV. After IAC had reached a maximum value, cell was superfused with ionomycin (10 µM). A: current records after IAC had reached a maximum value (top) and 12 min after superfusion of 10 µM ionomycin (bottom). B: IAC current amplitudes are plotted against time for same cell as in A. [Ca2+] of external solution was 2 mM.

Current-voltage relationships recorded before and after superfusion of ionomycin clearly showed that this ionophore selectively inhibited IAC. Furthermore, this inhibition was independent of voltage over a wide range of test potentials. In the experiment illustrated in Fig. 3, once IAC reached a stable amplitude, K+ currents were activated by voltage steps to test potentials between -50 and +60 mV before and after superfusion of ionomycin (10 µM). As shown in Fig. 3A, ionomycin selectively inhibited the IAC almost completely at each test potential, leaving only the IA (Fig. 3A, left and right). The effective inhibition of IAC by ionomycin at each test potential (Fig. 3B) indicated that ionomycin-stimulated Ca2+ influx did not merely shift the voltage dependence of IAC open probability to the right along the voltage axis.


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Fig. 3.   Voltage-independent inhibition of IAC by ionomycin. K+ currents were activated at 30-s intervals from a holding potential of -80 mV by voltage steps of various sizes before and after superfusion of cell with 10 µM ionomycin. A: current records at test potentials between -50 and +60 mV (in 10-mV increments) before (left) and after (right) superfusion of ionomycin. B: current-voltage relationships. IAC current amplitudes from experiment in A are plotted against test voltage [membrane potential (Vm)].

Experiments with ionomycin indicated that IAC could be specifically inhibited by raising [Ca2+]i through enhanced Ca2+ influx across the cell membrane. To further characterize the specific Ca2+ dependence of the response, we examined the effect of ionomycin on IAC when the Ca2+-buffering capacity of the pipette solution was increased by raising BAPTA in the pipette from 2 to 11 mM.

With the high-capacity Ca2+-buffering pipette solution, ionomycin was much less effective at inhibiting IAC. As illustrated in Fig. 4A, superfusion of this cell with ionomycin (10 µM) failed to reduce IAC, whereas ACTH (200 pM) completely inhibited this current within 3 min. ACTH inhibits IAC through a cAMP-dependent mechanism that appears to be independent of changes in [Ca2+]i (8). Overall, in four cells, ionomycin (10 µM) inhibited IAC by 25 ± 14% when the pipette contained 11 mM BAPTA, compared with 88 ± 4% (n = 14) inhibition observed with 2 mM BAPTA. In contrast, ACTH inhibition of IAC was insensitive to changes in pipette BAPTA concentration.


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Fig. 4.   Effect of intracellular Ca2+ buffering and external Ca2+ removal on ionomycin-mediated IAC inhibition. Inhibition of IAC by ionomycin (10 µM) was measured using high-capacity Ca2+-buffering pipette solution (11 mM BAPTA) and standard external solution (2 mM CaCl2; A) or external solution with no added Ca2+ (Ca2+ free) and pipette solution containing 2 mM BAPTA (B). Left: after IAC reached a stable amplitude, cells were sequentially superfused with ionomycin (10 µM) and adrenocorticotropic hormone (ACTH; 200 pM) while K+ currents were recorded. Middle: A-type K+ currents were inactivated either by a depolarizing prepulse (A) or by holding cell at -40 mV (B). Right: IAC current amplitudes are plotted against time. 1, maximum IAC; 2, IAC after ionomycin (10 µM); 3, IAC after ACTH (200 pM).

In other experiments, we found that with standard pipette solution (2 mM BAPTA) ionomycin failed to inhibit IAC when Ca2+ was omitted from the external solution. For Fig. 4B, the cell was superfused with external solution containing no added Ca2+ followed by a switch to solutions containing ionomycin (10 µM), followed by ACTH (200 pM). Ionomycin was ineffective in the absence of added Ca2+, whereas ACTH completely inhibited IAC.

Calmodulin and ionomycin inhibition of IAC. Many Ca2+-mediated processes, including the modulation of some ion channels, require calmodulin as an intermediate. Once activated by Ca2+, Ca2+/calmodulin may interact directly with the channel or indirectly through activation of a calmodulin-dependent enzyme. Calmodulin inhibitory peptide (residues 290-309 of calmodulin kinase II) potently inhibits calmodulin kinase II (IC50 50 nM) as well as other calmodulin-dependent processes (25). Calmodulin inhibitory peptide (2.5 µM), applied intracellularly through the recording pipette solution, failed to significantly alter ionomycin-mediated inhibition of IAC.

Figure 5 illustrates an experiment in which IAC was allowed to grow to a stable value in the presence of calmodulin inhibitory peptide before superfusion of ionomycin (10 µM). The inhibitory peptide had no apparent effect on IAC growth or inhibition by ionomycin. In this experiment, IAC was inhibited almost completely, but the IA was unaffected. Overall, with 2.5 µM calmodulin kinase II inhibitory peptide in the pipette solution, ionomycin (10 µM) inhibited IAC by 83 ± 7% (n = 8), compared with 88 ± 4% (n = 14) under control conditions. These results indicate that Ca2+ does not inhibit IAC through calmodulin acting either directly or through activation of a calmodulin-dependent enzyme.


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Fig. 5.   Effect of calmodulin (CaM) inhibitory peptide on ionomycin inhibition of IAC. Adrenal zona fasciculata cells (AZF cells) were voltage clamped in whole cell configuration using standard pipette solution supplemented with 2.5 µM CaM inhibitory peptide. After IAC had reached a stable amplitude, cells were superfused with ionomycin (10 µM) while IAC was recorded at 30-s intervals. A: K+ current records with (bottom) and without (top) depolarizing prepulses before (control) and after superfusion of ionomycin (10 µM). B: IAC amplitudes are plotted against time for experiment in A; open circle , with depolarizing prepulse; bullet , without prepulse.

AMP-PNP and ionomycin inhibition of IAC. The growth of IAC in whole cell recordings requires the presence of ATP at millimolar concentrations (6). Inhibition of this current by either ACTH or ANG II requires hydrolyzable forms of ATP, suggesting the involvement of a kinase or ATPase (6, 8, 22). To determine whether ATP hydrolysis is required for Ca2+-mediated inhibition of IAC, the nonhydrolyzable ATP analog AMP-PNP was substituted for ATP in the pipette solution. Figure 6 shows that, with 2 mM AMP-PNP in the pipette, ionomycin (10 µM) reversibly inhibited IAC by ~70%. Overall, ionomycin inhibited IAC by 75.4 ± 6% (n = 5) when AMP-PNP replaced ATP in the pipette. In each of three experiments, ACTH (200 pM) failed to produce any measurable inhibition of IAC when superfused before ionomycin, thus ensuring that AMP-PNP was present intracellularly at effective concentrations. The consistent inhibition of IAC by ionomycin with AMP-PNP in the pipette demonstrated that Ca2+ does not inhibit IAC by activation of an ATPase or Ca2+-dependent enzymes such as PKC.


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Fig. 6.   Effect of 5'-adenylylimidodiphosphate (AMP-PNP) on ionomycin-mediated inhibition of IAC. AZF cell was voltage clamped in whole cell configuration with pipette containing standard solution in which ATP was replaced with 2 mM AMP-PNP. After IAC had reached a stable amplitude, cell was superfused with ionomycin (10 µM) while K+ currents were recorded at 30-s intervals. A: K+ current records in response to voltage steps with (bottom) and without (top) depolarizing prepulses before (2) and after (3) superfusion of ionomycin. B: IAC amplitudes are plotted against time; open circle , with depolarizing prepulse; bullet , without prepulse. 1, immediately after initiation of whole cell recording; 2, after IAC had reached a maximum amplitude; 3, after inhibition by 10 µM ionomycin.

Effect of Ca2+ on unitary current. Whole cell patch-clamp experiments indicated that Ca2+-mediated inhibition of IAC might occur through a direct interaction of Ca2+ with the channel. Single-channel recording experiments in which Ca2+ was directly applied to the cytoplasmic surface of excised inside-out patches were consistent with this model of inhibition. In these experiments, the membrane patch was excised into an internal solution containing 22 nM Ca2+, and the holding potential was set to -40 mV (inside negative), a potential at which all IA channels are inactivated. Under these conditions, a single type of K+ current was typically present in the membrane patch. Figure 7A shows unitary currents recorded in response to voltage steps to +20 mV from a holding potential of -40 mV in control saline (20 nM Ca2+). Under these conditions, IAC channel activity increased spontaneously and continuously during prolonged recordings. Histogram analysis of unitary current amplitudes showed a major peak with a mean ± SE of 2.41 ± 0.82 pA, and two other peaks with means that were approximately twice (4.82 ± 0.73 pA) and three times (7.36 ± 0.67 pA) the unitary amplitude. When the cytoplasmic membrane surface was superfused with saline containing 35 µM Ca2+, IAC channel activity was dramatically reduced (Fig. 7B). Inhibition was evident after 30-60 s and reached a maximum within 2 min. When solution was switched back to low-Ca2+ saline, channel activity was restored to a level greater than control. Histogram analysis showed four separate peaks, each a multiple of the unitary amplitude (Fig. 7C). Unitary IAC were also inhibited by [Ca2+]i of 10 and 20 µM (data not shown).


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Fig. 7.   Inhibition of unitary IAC by Ca2+. Unitary IAC were recorded in inside-out configuration using pipettes containing standard external solution used in whole cell recordings (see METHODS). Patches were excised into internal solution containing 22 nM Ca2+. Voltage steps to +20 mV were applied at 30-s intervals from a holding potential of -40 mV. Amplitude histograms were constructed from unitary currents recorded in response to voltage steps 300 ms in duration. Current amplitudes were distributed into bins 0.150 pA in width. A-C: unitary currents (top) and corresponding amplitude histograms (bottom) from recordings in control solution (22 mM Ca2+), in presence of 35 µM Ca2+, and after return to control (wash), respectively. Currents were sampled at 5 kHz and filtered at a cutoff frequency of 2 kHz.

Effect of ionomycin on membrane potential. Results presented to this point indicate that IAC channels are inhibited by Ca2+ through a mechanism that is independent of calmodulin and protein kinases. Because IAC channels appear to set the membrane potential of AZF cells (8, 20), our findings suggest that increasing [Ca2+]i could trigger membrane depolarization through inhibition of these channels. To explore this possibility, we examined the effects of ionomycin on membrane potential and IAC in combined whole cell voltage-clamp and current-clamp recordings.

In the experiment illustrated in Fig. 8A, IAC was monitored in whole cell voltage clamp until it reached a stable value of ~325 pA (Fig. 8A, right). Membrane potential was then recorded after switching to current clamp. In control saline, the resting potential of the cell was -63 mV. The superfusion of ionomycin (10 µM) produced, after a delay of several minutes, a progressive depolarization that reached a maximum of ~45 mV during the next 2 min (Fig. 8A, left). At this time, voltage-clamp recordings showed that IAC had been inhibited by 90% but the IA was not affected (Fig. 8A, right). In a total of four similar experiments, ionomycin inhibited IAC by 89.6 ± 3.5% while depolarizing cells by an average of 47 ± 8 mV.


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Fig. 8.   Depolarization of AZF cells by ionomycin in low and high BAPTA. K+ currents were recorded at 30-s intervals with or without depolarizing prepulses. When IAC reached a stable maximum value, patch-clamp amplifier was switched to current clamp to record Vm. Patch pipettes contained either 2 mM (A) or 11 mM (B) BAPTA. Left: while Vm was recorded (sampling rate 10 s-1), cells were superfused with ionomycin or ACTH at indicated times. Right: K+ currents were recorded at times indicated on record of Vm by switching to voltage clamp. Traces were recorded with or without depolarizing prepulses as indicated by voltage protocol diagrams.

Ionomycin-mediated membrane depolarization was markedly reduced when the pipette contained the high-capacity Ca2+-buffering solution (11 mM BAPTA). In the experiment illustrated in Fig. 8B, ionomycin (10 µM) depolarized this cell by a maximum of <5 mV and failed to significantly inhibit IAC. When this same cell was then superfused with ACTH (200 pM), IAC was inhibited nearly completely and the cell was depolarized by ~40 mV. Overall, in recordings made with 11 mM BAPTA in the pipette, ionomycin (10 µM) inhibited IAC by 25.3 ± 14.6% (n = 4) and depolarized cells by only 8 ± 3 mV (n = 4).

Ca2+ and ANG II inhibition of IAC. In previous studies, we have shown that ANG II inhibits IAC by a maximum of ~75% under conditions in which [Ca2+]i was strongly buffered by 10 or 20 mM BAPTA (20, 22). Because ANG II inhibits IAC through activation of an AT1 receptor that is coupled to inositol 1,4,5-trisphosphate (IP3)-stimulated release of intracellular Ca2+, we tested the possibility that ANG II-mediated inhibition of IAC would be enhanced by reducing BAPTA in the patch pipette.

ANG II was significantly more effective at inhibiting IAC when [Ca2+]i was buffered with 2 mM rather than 11 mM BAPTA (Fig. 9). Overall, ANG II (10 nM) inhibited IAC by 76.5 ± 4.5% (n = 6) when [Ca2+]i was buffered to 0.02 µM with 11 mM BAPTA. In comparison, at the same concentration, ANG II inhibited IAC by 97.8 ± 3.4% (n = 4) when [Ca2+]i was buffered by 2 mM BAPTA (Fig. 9B).


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Fig. 9.   Effect of BAPTA on ANG II-mediated inhibition of IAC. Inhibition of IAC by ANG II (10 nM) was measured with pipette solutions containing 11 mM (A) or 2 mM (B) BAPTA. Top: K+ currents were activated by voltage steps to +20 mV, applied at 30-s intervals from a holding potential of -80 mV. 1, initial K+ current; 2, maximum current in control saline; 3, current after steady-state block by ANG II (10 nM). Bottom: IAC amplitude is plotted against time. Numbers correspond to traces at top.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Measures that increased [Ca2+]i in bovine AZF cells specifically inhibited IAC channel activity by a mechanism that appears to be independent of calmodulin, protein kinases, and ATP hydrolysis. The inhibition of IAC by the Ca2+ ionophore ionomycin was tightly linked to membrane depolarization. ANG II-mediated inhibition of IAC was blunted by strongly buffering [Ca2+]i with BAPTA. These results suggest a positive feedback mechanism by which ANG II-stimulated release of intracellular Ca2+ triggers IAC inhibition, membrane depolarization, and Ca2+ entry through voltage-gated Ca2+ channels.

Specificity and mechanism of IAC inhibition by Ca2+. The inhibition of IAC produced by raising [Ca2+]i, either by addition of Ca2+ to the patch electrode or through bath perfusion of ionomycin, was specific. IA was not altered. A direct action of ionomycin on IAC channels seems unlikely, since the effect of this ionophore was blunted or eliminated by removing external Ca2+ or by buffering [Ca2+]i strongly with 11 mM BAPTA.

Activation of IAC channels is weakly voltage dependent (6, 8). Ionomycin-mediated inhibition of IAC was independent of voltage over a wide range of test potentials. Presumably, Ca2+-mediated inhibition of IAC did not result from a rightward shift in the voltage dependence of IAC activation. In contrast, Ca2+ appears to act directly on Ca2+-activated K+ channels, producing a leftward shift in the voltage dependence of activation such that these K+ channels are activated at less-depolarized potentials (5). Apparently, Ca2+ modulation of these two K+ channel subtypes occurs through fundamentally different mechanisms.

Because the calmodulin inhibitory peptide, applied at 40 times its IC50, failed to alter ionomycin-mediated inhibition of IAC, it is unlikely that Ca2+-mediated inhibition occurs through activation of a calmodulin-dependent enzyme, such as calmodulin kinase or the Ca2+-dependent phosphatase calcineurin. Because this inhibitory peptide binds calmodulin with high affinity, it is also unlikely that Ca2+/calmodulin directly inhibits IAC channels (25).

The ability of ionomycin to inhibit IAC when ATP is replaced with the nonhydrolyzable ATP analog AMP-PNP indicates that Ca2+-mediated inhibition of IAC does not require the activation of any kinase or ATPase. Protein kinases typically require hydrolyzable forms of ATP at concentrations of <100 µM, whereas ATPases often display dissociation constants for this nucleotide in the millimolar range (11, 26).

The inability of calmodulin inhibitory peptide and AMP-PNP to prevent IAC inhibition by ionomycin suggests a direct interaction between Ca2+ and IAC channels. In this regard, IAC inhibition by ionomycin begins only after a delay of up to several minutes and requires additional minutes to reach a maximum. Although this would seem excessively long for a process requiring a direct interaction of Ca2+ with the channel, the impact of the combination of 10 µM ionomycin and 2 mM BAPTA on [Ca2+]i dynamics is unknown. It is clear that BAPTA is a rapidly acting Ca2+ buffer that can, at high concentrations, suppress [Ca2+]i increases induced by influx during action potentials or release from ligand-induced IP3 (14, 19, 30, 31). Furthermore, both ANG II- and ACTH-stimulated inhibition of IAC and membrane depolarization occur after a delay of one to several minutes, even in intact cells (20, 22). Thus the slow Ca2+-mediated inhibition could occur through a physiological mechanism instead of one generated as an artifact of the Ca2+-buffering system. In excised inside-out patches, inhibition of unitary current activity was somewhat faster but still required at least 2 min to reach a maximum.

Comparison with other Ca2+-inhibited ion channels. Ca2+ inhibits K+ channels in neurons and lymphocytes (1, 14, 15, 18, 29, 31). The mechanisms and signaling pathway that underlie Ca2+-mediated inhibition are varied. Im is a voltage- and time-dependent K+ current in neuronal cells whose inhibition by Ca2+ has been extensively studied. Raising [Ca2+]i to values >450 nM suppresses the expression of Im (31). Likewise, Im is inhibited by Ca2+ increases induced by muscarinic receptor activation or photolysis of a caged Ca2+ chelator, whereas inhibition is blunted by buffering Ca2+ with 20 mM BAPTA (14, 19, 31).

The inhibition of Im by Ca2+ may occur through a direct action on the K+ channel, since Ca2+ inhibits these channels in excised inside-out patches even in the absence of ATP (27, 28). However, it has been reported that Ca2+-dependent inhibition of Im in sympathetic neurons is mediated through activation of the Ca2+-dependent phosphatase calcineurin (18). It is unlikely that activation of a similar phosphatase is responsible for IAC inhibition, since calcineurin activation requires a calmodulin intermediate.

Other ion channels, including K+ channels in the kidney and cyclic nucleotide-gated channels of the olfactory epithelium, are modulated by Ca2+ through calmodulin-dependent mechanisms (4, 15). Inhibition of the cyclic nucleotide-gated channel by Ca2+ requires binding of the Ca2+/calmodulin complex to a specific site on the amino-terminal end of the channel (4, 17). IAC channels resemble cyclic nucleotide-gated cation channels in that the gating of each is regulated by cAMP (8, 13). However, Ca2+ modulation of IAC channels does not appear to occur through a calmodulin-dependent mechanism.

Physiological significance. Our findings indicate that membrane potential and [Ca2+]i are tightly coupled through the activity of IAC channels in AZF cells. Both ACTH and ANG II inhibit IAC, depolarize AZF cells and stimulate cortisol secretion. However, the importance of Ca2+ as an intracellular messenger linking peptide receptor activation to IAC inhibition has not been established.

ANG II stimulates cortisol secretion through the activation of a losartan-sensitive AT1 receptor. Although these AT1 receptors are coupled to PLC activation and IP3-stimulated release of Ca2+, ANG II-mediated inhibition of IAC may be mediated in part by a distinct Ca2+-independent signaling pathway (22). ANG II inhibits IAC by ~75%, even when [Ca2+]i is strongly buffered with 11 or 20 mM BAPTA (20, 22). Furthermore, under these conditions, ANG II-mediated inhibition of IAC requires the presence of hydrolyzable ATP (22). This result contrasts with ionomycin-mediated inhibition of IAC, which is not altered by substituting AMP-PNP for ATP.

The nearly complete inhibition of IAC by ANG II observed in the present study when pipette BAPTA was reduced to 2 mM is consistent with a model that includes activation of parallel signaling pathways, one of which utilizes Ca2+ as a mediator. Because nearly complete inhibition of IAC channels may be required to effectively depolarize AZF cells, separate inhibitory mechanisms that converge on IAC channels could provide an efficient mechanism for membrane depolarization.

In a previous study, we found that ANG II and ACTH produce nearly identical maximal depolarization of AZF cells as measured by high-resistance (100-150 MOmega ) intracellular electrodes (20). By comparison, ACTH was significantly more effective than ANG II at inhibiting IAC in whole cell patch-clamp experiments in which [Ca2+]i was strongly buffered with 11 mM BAPTA. This apparent discrepancy can now be explained if we assume that ANG II was more effective at increasing [Ca2+]i and inhibiting IAC in cells in which membrane potential was measured with sharp electrodes. In these intact cells, ANG II-mediated IAC inhibition would not be diminished by artificial Ca2+ buffering. Presumably, under these conditions, both ACTH and ANG II produce similar IAC inhibition and membrane depolarization.

cAMP is the primary intracellular messenger coupling ACTH receptor activation to membrane depolarization (8). Accordingly, ACTH produces nearly complete inhibition of IAC even when [Ca2+]i is strongly buffered with 11 mM BAPTA. However, Ca2+ entering through low-voltage-activated T-type channels in these AZF cells could contribute to IAC inhibition. Specifically, at low ACTH concentrations, partial inhibition of IAC channels mediated by cAMP could produce sufficient depolarization to activate T-type Ca2+ channels, producing further IAC inhibition and membrane depolarization (21).

The inhibition of IAC channels by Ca2+ at physiological concentrations identifies a novel mechanism whereby AZF cell membrane potential is tightly linked in a reciprocal relationship to [Ca2+]i. Agents that trigger Ca2+ increases of sufficient magnitude by influx across the cell membrane or release from intracellular stores would inevitably depolarize AZF cells. This system incorporates the elements of a positive feedback mechanism driven by a regenerative Ca2+ signal. In addition to inducing the synthesis of corticosteroids in these secretory cells, [Ca2+]i would, at the same time, regulate its own entry across the plasma membrane (3, 7, 24).

The IAC channel is a distinctive new type of K+ channel with properties that identify it as a central control point for the regulation of cortisol secretion. In addition to setting the resting potential of AZF cells, IAC channel activity is regulated by a variety of metabolic factors and hormonally induced second messengers. Although IAC open probability is greatly enhanced by ATP at physiological concentrations, it is inhibited by peptide hormone-generated second messengers including cAMP and Ca2+. Thus IAC channels are intracellular sensors that integrate complex metabolic and hormonal signals, coupling the metabolic state of the cell to membrane potential and cortisol secretion.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47875 and by National American Heart Association Grant-in-Aid 94011740 to J. J. Enyeart.

    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: J. J. Enyeart, Dept. of Pharmacology, Ohio State University College of Medicine, 5188 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239.

Received 13 April 1998; accepted in final form 19 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(6):C1526-C1537
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