TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells

Judith A. Enyeart,1 Sanjay J. Danthi,1,2 and John J. Enyeart1

1Department of Neuroscience, College of Medicine and Public Health, and 2Mathematical Biosciences Institute, The Ohio State University, Columbus, Ohio 43210

Submitted 27 May 2004 ; accepted in final form 9 August 2004


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 ABSTRACT
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Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K+ channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K+ channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K+ current and a noninactivating background K+ current with properties that collectively identify it as bTREK-1. The outwardly rectifying K+ current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1–3,4-dihydroxy-{alpha}-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K+ current by 82.1 ± 6.1% and depolarized AZG cells by 31.6 ± 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K+ current by 73.8 ± 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca2+ current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca2+-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K+ channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca2+ entry and aldosterone secretion.

patch clamp; two-pore K+ channel


ANGIOTENSIN II (ANG II) is a principal physiological stimulus for aldosterone secretion by bovine adrenal glomerulosa (AZG) cells (3, 41). Although ANG II-stimulated aldosterone secretion is mediated through the activation of a losartan-sensitive AT1 receptor, the specific signaling pathways involved are only partially understood. In particular, the roles of specific ion channels and depolarization-dependent Ca2+ entry in the process have not been clarified. In this regard, both bovine and rat AZG cells maintain negative resting potentials and express both voltage-gated T- and L-type Ca2+ channels (26, 41, 46), as well as voltage-gated and background K+ channels (4, 10, 25, 29, 31, 43).

ANG II-stimulated aldosterone secretion depends, at least in part, on Ca2+ entry through voltage-gated T- and L-type Ca2+ channels (6, 24, 25, 46). Several studies indicate that ANG II enhances the activity of low voltage-activated T-type Ca2+ channels in AZG cells (6, 9, 26, 31). The enhanced activity of T-type Ca2+ channels was associated with an ~10-mV negative shift in the voltage dependence of T channel activation (6, 31). These actions of ANG II may occur through activation of an AT1 receptor through a mechanism that involves calmodulin-dependent protein kinase II (1, 27).

In contrast to the above findings, other patch clamp studies on rat and bovine AZG cells reported that ANG II either has no effect or actually inhibits T-type Ca2+ channels in these cells (25, 45). Furthermore, ANG II does not directly activate high voltage-activated L-type Ca2+ channels in AZG cells (24, 25, 30). Overall, it is unlikely that ANG II enhances Ca2+ entry in AZG cells solely through a direct action on voltage-gated Ca2+ channels.

Other studies reported that ANG II depolarizes murine, feline, bovine, and human AZG cells by inhibiting unidentified background K+ channels, thereby suggesting a specific mechanism for the indirect activation of voltage-gated Ca2+ channels (4, 25, 41, 43). However, until recently, K+ channels that could set the resting potential of AZG cells and whose inhibition by ANG II would be coupled to depolarization-dependent Ca2+ entry have not been identified. In recent years, more than one dozen two-pore/four-transmembrane (2P/4TMS) background K+ channels have been identified. These background K+ channels exhibit little voltage dependence, remain open at negative membrane potentials, and set the resting potential of a wide range of cells (19, 39). Recently, rat AZG cells were shown to express the 2P/4TMS K+ channels TASK-1 and TASK-3 (10, 11). TASK-3 was reported to be the dominant background K+ channel in these cells (10). However, neither TASK-1 nor TASK-3 was shown to set the resting potential of AZG cells, nor was inhibition of either channel by ANG II shown to mediate membrane depolarization.

Cortisol-secreting bovine adrenal zona fasciculata (AZF) cells express bTREK-1 background channels that are inhibited through activation of multiple native G protein-coupled receptors. These include receptors for the peptides ACTH and ANG II, as well as P2Y nucleotide and multiple P1 adenosine receptors (16, 32, 33, 51, 52). Inhibition of bTREK-1 through all of these receptors is coupled to AZF cell depolarization.

We now report that bovine AZG cells also robustly express bTREK-1 K+ channels and that, in these cells, they set the resting membrane potential. Inhibition of these channels by ANG II is tightly coupled to membrane depolarization. Specific activators of bTREK-1 channels reverse ANG II-mediated depolarization and suppress aldosterone secretion. Under conditions wherein ANG II produced nearly complete inhibition of bTREK-1, this peptide had no effect on the T-type Ca2+ current.


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 MATERIALS AND METHODS
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Materials

Tissue culture media, antibiotics, fibronectin, and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA). Coverslips were from Bellco (Vineland, NJ). Phosphate-buffered saline (PBS), enzymes, 1,2,-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ATP, arachidonic acid (AA), AMP-PNP, and ANG II were from Sigma (St. Louis, MO). Baicalein and cinnamyl 1–3,4-dihydroxy-{alpha}-cyanocinnamate (CDC) were obtained from Biomol (Plymouth Meeting, PA). rTASK-3 cDNA was the kind gift from both Dr. D. Kim (Department of Physiology and Biophysics, Finch University of Health Sciences/The Chicago Medical School) and Dr. R. Preisig-Müller (Institut für Normale und Pathologische Physiologie, Marburg University). Marathon-ready cDNA from normal human adrenals was obtained from Clontech (Palo Alto, CA).

Methods

Isolation and culture of AZG cells. Bovine adrenal glands were obtained from steers (age range 2–3 yr) within 1 h 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 AZG cells were obtained and prepared as previously described (40), with some modifications. Briefly, glomerulosa cells were isolated from adrenal capsular tissue and cells adherent to the capsule. Capsular tissue was cut into small (0.2–0.5 cm2) pieces. Tissue was digested for 1 h at 37°C in DMEM-F12 (1:1) with dispase (10 mg/ml), BSA (1%, wt/vol) and 50 µg/ml DNase. After digestion, the tissue suspension was strained through two layers of cheesecloth, and cells were either resuspended in DMEM-F12 (1:1) with 10% FBS, 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 FBS-5% DMSO, divided into aliquots, and stored in liquid nitrogen for future use. Cells were plated in 35-mm dishes for secretion experiments or 35-mm dishes containing 9-mm2 glass coverslips for electrophysiology experiments. Dishes or coverslips were treated with fibronectin (10 µg/ml) at 37°C for 30 min and then rinsed with warm, sterile PBS immediately before cells were added. Cells were plated in DMEM-F12+ and were maintained at 37°C in a humidified atmosphere of 95% air-5% CO2.

Patch clamp experiments. Patch clamp recordings of K+ channel currents were made in the whole cell configuration. The standard pipette solution consisted of 120 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 11 mM BAPTA, 10 mM HEPES, 1 mM ATP, and 200 µM GTP, with pH titrated to 7.1 with KOH. Pipette solution of this composition yielded a free Ca2+ concentration of 2.2 x 10–8 M, as determined by the Bound and Determined software program (5). In some experiments, MgATP in the pipette solution was raised to 5 mM and pH lowered to 6.4, as noted in the text. The external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 5 mM glucose, with pH adjusted to 7.4 with NaOH. All solutions were filtered through 0.22-µm cellulose acetate filters.

Recording conditions and electronics. AZG cells were used for patch clamp experiments 2–12 h after plating. Because AZG cells are significantly smaller than AZF cells, cells with capacitances of 7–12 pF were selected for recording. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume 1.5 ml), which was continuously perfused by gravity as a rate of 3–5 ml/min. Patch electrodes with resistances of 2–3 M{Omega} were fabricated from Corning 0010 glass (World Precision Instruments, Sarasota, FL). These electrodes routinely yielded access resistances of 1.5–5.0 M{Omega} and voltage clamp time constants of <100 µs. K+ currents were recorded at room temperature (22–25°C) according to the procedure of Hamill et al. (22) by use of a List EPC-7 patch clamp amplifier.

Pulse generation and data acquisition were done using a personal computer and PCLAMP software with TL-1 interface (Axon Instruments, Burlingame, CA). Currents were digitized at 2–10 KHz after filtering with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records by use of summed scaled hyperpolarizing steps of one-third to one-fourth pulse amplitude. Data were analyzed using PCLAMP (CLAMPFIT 6.04) and SigmaPlot (version 8.0) software. Drugs were applied by bath perfusion, controlled manually by a six-way rotary valve.

Measurement of bTREK-1 K+ currents. The absence of time- and voltage-dependent inactivation of the bTREK-1 K+ current allowed it to be easily isolated for measurement in whole cell recordings from AZG cells, using either of two voltage clamp protocols. When voltage steps of 300-ms duration were applied from a holding potential of –80 mV to a test potential of +20 mV, bTREK-1 could be selectively measured near the end of the voltage step, where the rapidly inactivating bKv1.4 K+ current had completely inactivated. Alternatively, bTREK-1 was selectively activated with an identical voltage step, after a 10-s prepulse to –20 mV had fully inactivated bKv1.4 K+ current (see Fig. 2A).



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Fig. 2. Activation of a noninactivating K+ current in AZG cells by ATP and acidification of the pipette solution. Whole cell K+ currents were recorded from AZG cells with pipette solutions containing 5 mM ATP at pH 6.4 or 1 mM MgATP at pH 7.1. Currents were activated by voltage steps to +20 mV with or without depolarizing prepulses, as illustrated and described in Methods. Current traces show that K+ currents recorded after the noninactivating current had reached maximum value. K+ current amplitudes are plotted against time at right for recordings made with and without depolarizing prepulses. Nos. on traces correspond to those on graph.

 
Aldosterone secretion experiments. AZG cells were cultured on fibronectin-coated 35-mm dishes at a density of 1.5 x 106 cells/dish in defined media [DMEM-F12 (1:1), 50 µg/ml BSA, 100 µM ascorbic acid, 1 µM tocopherol, 10 nM insulin, and 10 µg/ml transferrin]. After 1 h, the medium was aspirated and changed to defined medium (DMEM-F12 with 50 µg/ml BSA, 100 µM ascorbic acid, 1 µM tocopherol, 0.15 µg/ml insulin, and 10 µg/ml transferrin) either without (control), or with CDC or ANG II or these two in combination. Drugs were added directly to media in dishes from concentrated stock. Samples (200 µl) of media were collected at selected times and frozen at –20°C for later assay. Aldosterone concentration was determined using a solid-phase radioimmunoassay kit (Diagnostic Products, Los Angeles, CA). Experiments were performed in triplicate and assayed for aldosterone in duplicate.

Northern blot analysis. RNeasy columns (Qiagen, Valencia, CA) that had been treated with RNase-free DNase (Qiagen) to remove genomic contamination were used to extract total RNA from AZG and AZF cells that had been cultured in DMEM-F12+ for 8 h.

Total RNA was separated on a denaturing 8% formaldehyde-1.0% agarose gel and transferred to a nylon membrane (Gene Screen Plus, NEN). RNA was fixed to the membrane by UV cross-linking using a Stratalinker (Stratagene, La Jolla, CA). Northern blot was prehybridized in a heat-sealable plastic bag for 2 h at 42°C in ULTRAhyb (Ambion, Austin, TX) and then hybridized with a random-primed [{alpha}-32P]dCTP radiolabeled 379-bp EcoRI fragment of bTREK-1 or a full-length TASK-3 cDNA (Prime-it-II, Stratagene) overnight in minimal volume of hybridization solution at 42°C. After 18–24 h, blots were washed twice at room temperature in 2x SSPE for 15 min, twice at 40°C in 1x SSPE, and 1% SDS for 30 min. For TREK-1 hybridization, a more stringent wash, twice at 65°C with 0.1x SSPE, and 1% SDS for 15 min was necessary. Autoradiograms were obtained by exposing blots to Kodak X-O-Mat AR film at –70°C for 1 h (bTREK-1) or 24 h (TASK-3).

In situ hybridization.
TISSUE PREPARATION. Bovine tissue, obtained as described above, was immersed in 4% paraformaldehyde-0.1 M sodium phosphate buffer, pH 7.4, at 4°C for 1–3 h to preserve morphology and then embedded in OCT (Tissue-Tek) embedding matrix for frozen sectioning in embedding molds. Frozen blocks were allowed to equilibrate with cryostat chamber at –7°C. Tissue was cut into 10-µm sections and then thaw-mounted onto charged slides (SuperFrost/Plus). Slides were stored at –80°C. Before hybridization, slides were allowed to equilibrate to room temperature and then fixed in a 4% paraformaldehyde solution. Slides were then subjected to a series of washes in 0.1 M PBS (pH 7.4) and acetic anhydride (0.25% I 0.1 M triethanolamine, 0.9% NaCl, pH 8.0) and subsequently dehydrated and delipidated in a series of ethanol washes. The hybridization reaction was carried out overnight at 37°C in the presence of 60 µl of hybridization buffer (Amresco) with 3'-terminal 35S-labeled oligo, 100 mM DTT, and 250 µg/ml yeast tRNA. Slides were washed in SSC of increasing stringency, dehydrated by a series of ethanol washes, and then exposed to Biomax film (Kodak) for 3 days to evaluate signal.


OLIGONUCLEOTIDE PROBES. A bTREK-1 probe was designed using template region bp 1167–1203, where there is much less sequence similarity to other 2P/4TMS channels such as TREK-2, TASK, or TRAAK. A sense probe was used to assay nonspecific binding. A bovine 11{beta}-hydroxylase (CYP11B) 32-nt oligo (5'-GTC CAG CTG GGA TGT GGT AGT TCT GCA GCA CC-3') was used as a positive control to provide morphological identification of adrenal tissue regions. Specific bTREK-1 probe sequences were as follows: antisense: 5'-CTT GTC ATA AAT CTC CAC GCT CAG CCG CCT CCT GGT T-3' (37 nt), and sense control: 5'-aac cag gag gcg gct gag cgt gga gat tta tga cca g-3'(37 nt). Oligos were synthesized and PAGE purified by IDT (Coralville, IA). Probe sequences were checked against sequences in GenBank to ensure no cross-reactivity with other two-pore K+ channel gene products or sequences in the database.


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bTREK-1 mRNA Expression in Bovine Adrenal Cells

Northern blot analysis was used to characterize the relative expression of mRNA coding for TREK-1 and TASK-3 in bovine AZF and AZG cells. Figure 1A shows that AZF and AZG cells both express bTREK-1 mRNA, which is present in three transcripts of 4.9, 3.6, and 2.8 kb. Furthermore, as previously shown in AZF cells, bTREK-1 mRNA is also markedly induced in AZG cells by a 20-h treatment with forskolin (5 µM) (14).



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Fig. 1. Distribution of bovine (b)TREK-1 and TASK-3 mRNA expression in bovine adrenal gland, determined by Northern blot and in situ hybridization. A: Northern blot analysis. Bovine adrenal zona fasciculata (AZF; F) and adrenal glomerulosa (AZG; G) cells were cultured as described in Methods for 8 h before medium was replaced with the same medium with or without forskolin (FORSK.,5 µM) as indicated for 20 h before isolation of RNA. For Northern blots, 10 µg of total AZG or AZF RNA isolated from control or forskolin-treated cells were loaded in duplicate lanes. The membrane was divided into similar lanes and hybridized as described in Methods with either a 370-bp EcoRI fragment from the 5' end of bTREK-1 or the full-length cDNA for rat (r)TASK-3. B: in situ hybridization. mRNA for bTREK-1 and 11{beta}-hydroxylase (CYP11B) was detected in bovine adrenal cross sections by hybridization with 35S-labeled oligonucleotide probes, as described in Methods. Top 1: specific binding of labeled oligonucleotide probe (antisense) to bTREK-1 mRNA transcript. Top 2: x3 magnification of highlighted area in Top 1. Top 3: hybridization with bTREK-1 sense oligonucleotide probe to show nonspecific binding. Bottom 1: for morphological identification, binding of an oligonucleotide probe specific to CYP11B mRNA known to be present in both glomerulosa and fasciculata is shown. Bottom 2: x3 magnification of highlighted area in Bottom 1. Bottom 3: hybridization with CYP11B sense oligonucleotide probe to show nonspecific binding. Scale bar (5 mm) applies to columns 1 and 3.

 
In contrast to TREK-1, TASK-3 mRNA was poorly expressed in these same bovine AZF and AZG cells. TASK-3 was undetectable in the RNA from both control and forskolin-treated cells after the film was exposed for 24 h, whereas bTREK-1 mRNA was easily detected with a 1-h exposure.

In situ hybridization experiments confirmed the finding that bTREK-1 mRNA is robustly expressed throughout the bovine adrenal cortex, including the glomerulosa and fasciculata. These experiments also indicated that bTREK-1 is undetectable in the adrenal medulla. The experiment illustrated in Fig. 1B shows that mRNAs for bTREK-1 and CYP11B, a steroid hydroxylase in the pathways that convert cholesterol to cortisol and aldosterone (35), are both strongly expressed in bovine AZF and AZG tissue, but not in the adrenal medulla. In this regard, no distinction could be made between the level of bTREK-1 expression in the subcapsular glomerulosa and the adjacent fasciculata. The presence of bTREK-1 mRNA in AZF as well as AZG cells suggests that the corresponding TREK-1 K+ channels are expressed in glomerulosa cells.

bTREK-1 K+ Channels are Expressed in Bovine AZG Cells

AZG cells were isolated for patch clamp recording, and K+ currents from these cells were activated by two different voltage clamp protocols, as described in Methods. Whole cell patch clamp recordings showed that bovine AZG cells uniformly expressed two types of K+ currents that were similar to those previously described in bovine AZF cells (16, 17, 34). These included a rapidly inactivating A-type K+ current and a noninactivating K+ current with a large instantaneous and a smaller time-dependent component.

bTREK-1 is distinctive among 2P/4TMS channels in its activation by ATP and intracellular acidification (15, 16, 53). The noninactivating K+ current in AZG cells was activated by both ATP and low pH. In the experiment illustrated in Fig. 2, whole cell K+ currents were recorded with pipette solutions containing 5 mM MgATP at pH 6.4 or 1 mM MgATP at pH 7.1. When recordings were made with 5 mM MgATP at pH 6.4, the noninactivating K+ current spontaneously increased in amplitude for a period of minutes before it reached a stable maximum value (Fig. 2). In contrast, when the K+ current was recorded at pH 7.1 with 1 mM MgATP in the pipette, the noninactivating current was less prominent and failed to grow significantly beyond its initial amplitude. Overall, with acidified pipette solution containing 5 mM MgATP, the putative bTREK-1 reached a maximum current density of 27.9 ± 4.8 pA/pF (n = 24). By comparison with standard pipette solution, this current reached a maximum current density of only 11.1 ± 2.2 pA/pF (n = 12).

In situ hybridization experiments showed that bovine adrenal chromaffin cells express little or no bTREK-1 mRNA. Accordingly, bTREK-1 current was undetectable in whole cell patch clamp recordings from enzymatically dissociated chromaffin cells with acidified pipette solution containing 5 mM ATP. In recordings from eight cells, only voltage-gated K+ currents similar to those previously described were observed (28) (data not shown).

AA Activates the Background K+ Current in AZG Cells

Although the noninactivating current enhanced by acidified pipette solution containing 5 mM MgATP resembled that of bTREK-1, additional evidence was needed to establish its identity. Of the more than one dozen background K+ channels characterized thus far, only the mechanogated subgroup including TREK-1, TREK-2, and TRAAK is activated by AA and other polyunsaturated fatty acids (12, 18, 19, 39). In AZG cells, AA triggered a pronounced increase in the amplitude of a noninactivating current with voltage-dependent rectification indistinguishable from bTREK-1 (16).

In the experiment illustrated in Fig. 3A, whole cell K+ currents were activated with and without depolarizing prepulses, using pipettes containing standard internal solution (pH 7.1, 1 mM MgATP). After the noninactivating current reached a stable amplitude, the cell was superfused with 10 µM AA, which increased this K+ current more than 17-fold within 8 min, whereas the rapidly inactivating A-type current was completely inhibited.



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Fig. 3. Arachidonic acid (AA) activates the noninactivating outwardly rectifying K+ current of AZG cells. Whole cell K+ currents were recorded from AZG cells while control saline or saline containing 10 µM AA was superfused. A: temporal pattern and reversibility. K+ currents were recorded from an AZG cell at 30-s intervals in response to voltage steps to +20 mV from a holding potential of –80 mV with ({circ}) or without ({bullet}) a depolarizing prepulse, as indicated. After recording of currents in standard saline, cell was superfused with 10 µM AA for 10 min. Nos. on traces correspond to those on plot of current amplitudes at right. B: voltage-dependent rectification of K+ current activated by acidified pipette solution containing 5 mM MgATP and externally applied AA. Linear voltage ramps of 100 mV/s were applied from a holding potential of 0 mV to potentials between +60 and –140 mV with pipette solutions containing 5 mM MgATP at pH 6.4 (control). A second voltage ramp was applied after AA (10 µM) produced maximum increase in noninactivating current. C: bar graphs summarize data from experiments as in Figs. 1 and 2A. Values are means ± SE of indicated no. of determinations.

 
The increase in amplitude of the noninactivating K+ current was rapidly reversed, whereas inhibition of the A-type K+ current was poorly reversible upon superfusion of control saline (Fig. 3, A and B). Overall, 10 µM AA increased the noninactivating current density of six AZG cells from 6.7 ± 2.0 to 162.4 ± 28.5 pA/pF (Fig. 3C).

AA also markedly increased the noninactivating K+ current in cells where the current had been preactivated with acidified pipette solution containing 5 mM MgATP. Although the noninactivating K+ current reached a maximum density of 22.0 ± 3.8 pA/pF (n = 6) in control saline, it grew to 222.0 ± 50.5 pA/pF (n = 6) in the presence of 10 µM AA (Fig. 3C).

In the presence of standard external solution, bTREK-1 appears as an outwardly rectifying K+ current (12, 16, 53). The voltage-dependent rectification of the background K+ current in AZG cells activated by ATP and acidified pipette solution and by 10 µM AA was characterized using voltage ramps. In the experiment illustrated in Fig. 3B, K+ currents were recorded with acidified pipette solution (pH 6.4) containing 5 mM MgATP before and after the cell was superfused with 10 µM AA. Under bath conditions, linear voltage ramps applied between +60 and –140 mV induced similar outwardly rectifying currents that reversed at potentials near the theoretical K+ equilibrium potential. Thus AZG cells express a background K+-selective current with properties indistinguishable from those of bTREK-1.

ANG II Inhibits bTREK-1 and Depolarizes AZG Cells

In situ hybridization, Northern blot, and patch clamp experiments indicate that bTREK-1 is the predominant background K+ channel expressed by bovine AZG cells. If bTREK-1 K+ channels set the resting membrane potential, then inhibition of these channels by ANG II should be coupled to depolarization.

In whole cell recordings, ANG II (10 nM) potently and selectively inhibited the noninactivating K+ current. Inhibition began within 90 s and typically reached a steady-state value in 5–7 min (Fig. 4A). Overall, ANG II (10 nM) inhibited bTREK-1 current by 82.1 ± 6.1% (n = 7; Fig. 4, A and C).



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Fig. 4. Angiotensin (ANG) II inhibits acid- and ATP-activated background K+ current and depolarizes AZG cells. A: whole cell K+ currents were activated by voltage steps to +20 mV applied at 30-s intervals from a holding potential of –80 mV with ({circ}) or without ({bullet}) depolarizing prepulses to –20 mV, as indicated. After noninactivating K+ current reached stable maximum value, cell was superfused with 10 nM ANG II. bTREK-1 amplitude is plotted against time at right. Nos. on traces correspond to those on graph. B: ANG II depolarization of AZG cells. Membrane potential (Vm) was recorded in current clamp with pipette solution containing 5 mM MgATP at pH 6.4. After 4 min, cell was superfused with saline containing 10 nM ANG II, as indicated. Vm was sampled at 100-ms intervals. Each data point is the average of 100 values sampled over a 10-s interval. C: inhibition of bTREK-1 by ANG II and vasopressin. Summary of experiments in which AZG cells were superfused with either ANG II (2 nM) or vasopressin (50 nM) and inhibition of bTREK-1 was measured. Values are means ± SE of indicated no. of determinations.

 
In current clamp recordings of AZG cell membrane potential, it was discovered that ANG II-mediated inhibition of bTREK-1 current was accompanied by membrane depolarization. In the experiment illustrated in Fig. 4B, ANG II (10 nM) depolarized this AZG cell by 30 mV from its resting value of –58 mV. Maximum depolarization occurred within 6 min. Overall, in current clamp recordings, AZG cells maintained a resting membrane potential of –63.6 ± 2.3 mV (n = 5). ANG II (10 nM) depolarized these cells by an average of 31.6 ± 2.3 mV with a temporal pattern that paralleled bTREK-1 inhibition.

Vasopressin Inhibits bTREK-1 Current in AZG Cells

If ANG II-stimulated secretion is mediated through bTREK-1 inhibition, then other agents that physiologically induce aldosterone secretion might also inhibit this background K+ current. Vasopressin stimulates aldosterone secretion through a phospholipase C-coupled receptor (48). Vasopressin (50 nM) inhibited bTREK-1 in AZG cells by an average of 73.8 ± 9.4% (n = 4; Fig. 4C).

AA Overwhelms ANG II-Mediated Inhibition of bTREK-1 and Hyperpolarizes AZG Cells

The correlation that exists between ANG II-mediated inhibition of bTREK-1 and membrane depolarization provides further evidence that these channels act pivotally in setting the membrane potential of AZG cells. If so, then activation of bTREK-1 channels by agents such as AA should oppose membrane depolarization by ANG II.

In whole cell patch clamp experiments, it was discovered that AA overwhelmed the inhibition of bTREK-1 by ANG II and completely reversed ANG II-mediated membrane depolarization. In the experiment illustrated in Fig. 5, ANG II (2 nM) produced nearly complete inhibition of bTREK-1 (Fig. 5A, trace 2) and depolarized the cell by 28.6 mV from its resting potential of –68.6 mV (Fig. 5B). Superfusion of the cell with saline containing 2 nM ANG II and 10 µM AA dramatically increased bTREK-1 to an amplitude nearly six times the control value (Fig. 5A, trace 3). This increase in bTREK-1 was accompanied by a rapid hyperpolarization from –40.6 to –72.7 mV within 2 min (Fig. 5B).



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Fig. 5. AA reverses ANG II-mediated inhibition of bTREK-1 and membrane depolarization. AZG cell was voltage clamped in whole cell mode with pipette solution containing 5 mM MgATP at pH 6.4. Whole cell K+ currents were recorded in response to voltage steps to +20 mV from a holding potential of –80 mV. After bTREK-1 reached maximum value, Vm was recorded under current clamp. While Vm was being recorded, cell was superfused sequentially with saline containing ANG II (2 nM) and then with ANG II + AA (10 µM). bTREK-1 current was recorded intermittently by switching to voltage clamp. A: bTREK-1 currents recorded at times indicated by nos. in B. B: AZG membrane potential was sampled at 100-ms intervals and plotted as averaged values obtained over 10-s intervals. Nos. on plot correspond to those on current traces in A.

 
CDC Activates bTREK-1, Reverses ANG II-Stimulated Membrane Depolarization, and Inhibits Aldosterone Secretion

Recently, we demonstrated that CDC and other selected caffeic acid esters markedly enhance the activity of native AZF cell and cloned bTREK-1 channels (13). CDC also significantly increased the noninactivating K+ current in AZG cells. In the experiment illustrated in Fig. 6A, CDC (20 µM) increased the noninactivating K+ current more than 17-fold in 8 min before the gigohm seal was lost. Overall, CDC (10 or 20 µM) increased this current density in six AZG cells from a control value of 15.6 ± 3.1 to 192.6 ± 65.5 pA/pF (n = 6).



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Fig. 6. Cinnamyl 1-3,4-dihydroxy-{alpha}-cyanocinnamate (CDC) activates bTREK-1 and reverses ANG II-mediated depolarization of AZG cells. A: CDC activates bTREK-1 in AZG cells. Whole cell K+ currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of –80 mV, using standard pipette solution (1 mM MgATP, pH 7.1). Cell was superfused with 20 µM CDC as indicated. Current amplitudes are plotted against times at right. Nos. on plot correspond to current traces. B: CDC reverses ANG II depolarization. AZG membrane potential was recorded in current clamp. Cell was sequentially superfused with saline containing ANG II (2 nM) followed by ANG II + CDC (20 µM). Membrane potential was sampled at 100-ms intervals and plotted as averaged values over 10-s intervals.

 
CDC resembles AA in effectively opening bTREK-1 K+ channels in AZG cells. Within the framework of our model, CDC would also be expected to reverse ANG II-stimulated depolarization. In the experiment illustrated in Fig. 6B, ANG II depolarized an AZG cell by 30.0 mV from its resting potential of –63.2 mV. Superfusing the cell with CDC (20 µM) repolarized the cell within 9 min from –33.2 to –58.0 mV. Similar results were obtained in each of three cells.

Because CDC reverses ANG II-stimulated membrane depolarization, it should also inhibit depolarization-dependent aldosterone secretion. In the experiment illustrated in Fig. 7, CDC (20 µM) inhibited ANG II-stimulated aldosterone secretion measured at 1.5 and 16 h by 83 and 95.4%, respectively. CDC also inhibited unstimulated aldosterone secretion at 1.5 and 16 h by 32 and 56.8%, respectively, in the same experiment. Overall, at 1.5 h in three separate experiments, CDC inhibited ANG II-stimulated and unstimulated aldosterone secretion by 70.5 ± 7.6 and 39.7 ± 4.4%. The inhibitory effects of CDC on aldosterone secretion were reversible, and this drug did not affect cell viability as determined by Trypan blue exclusion (data not shown).



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Fig. 7. CDC inhibits ANG II-stimulated aldosterone secretion. Cultured AZG cells were incubated in serum-free defined medium (see Methods) or the same medium containing ANG II (10 nM), CDC (20 µM), or ANG II (10 nM) and either CDC (20 µM) or baicalein (10 µM). Medium samples were collected at 1.5 and 16 h and assayed for aldosterone as described in Methods. Values are means ± SE of indicated no. of determinations.

 
It has been reported that ANG II-stimulated aldosterone secretion is mediated, in part, by 12-lipoxygenase products of AA (20, 36). Because CDC is a potent 12-lipoxygenase antagonist (IC50 = 0.06 µM), the possibility that CDC-mediated inhibition of aldosterone secretion occurs through inhibition of this enzyme, rather than bTREK-1 activation, had not been eliminated (8).

Baicalein inhibits 12-lipoxygenase with an IC50 of 0.015 µM but does not activate bTREK-1 (8, 13). At a concentration of 10 µM, baicalein failed to inhibit ANG II-stimulated aldosterone secretion at either 1.5 or 16 h (Fig. 7).

ANG II Has No Effect on IT-Ca in AZG Cells

ANG II inhibited bTREK-1 currents in AZG cells with pipette solutions containing 5 mM MgATP and intracellular Ca2+ concentration ([Ca2+]i) buffered to 22 nM using 11 mM BAPTA. Experiments were done to determine whether ANG II modulated voltage-gated Ca2+ currents through the same signaling pathway.

In whole cell recordings of Ca2+ current, the majority of AZG cells express only low voltage-activated T-type Ca2+ currents that are distinguished by their rapid inactivation and slow deactivation kinetics (Fig. 8A). The slow rate of T channel closing is observed in whole cell recordings as a prominent, decaying "tail" current upon repolarization after a brief depolarizing step (Fig. 8A, right traces).



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Fig. 8. Effect of ANG II on T-type Ca2+ current (IT-Ca). A: IT-Ca were activated by long (300 ms) test pulses to –5 mV (left traces) or short (10 ms) test pulses to 0 mV (right traces) applied at 30-s intervals from a holding potential of –80 mV. Representative traces from control (black) and ANG II (2 nM)-treated cells (red) (left traces) are superimposed. For tail currents (right), traces from control (black) and ANG II (10 nM)-treated cells (blue) are superimposed. Peak current amplitudes for both protocols are plotted against time in graph. B: ANG II and the IV relationship. IT-Ca were activated from –80 mV by voltage steps applied at 0.1 Hz to various test potentials between –60 and +50 mV before and after superfusion of 2 nM ANG II. Representative current traces recorded at indicated test potentials (–30, –20, and –10 mV) before (black) and after (red) superfusion of 2 nM ANG II are superimposed. Current-voltage relationship: peak current amplitudes from 3 cells before ({circ}, black line) and after ({bullet}, red line) addition of 2 nM ANG II were averaged and plotted against test potential. Values are means ± SE.

 
The modulation of IT-Ca in AZG cells by ANG II was monitored in whole cell recordings with pipette solutions containing nucleotides and 11 mM BAPTA to buffer Ca2+, as described above for recording K+ currents. Under these conditions, ANG II (2 or 10 nM) failed to alter IT-Ca amplitudes measured in response to short (10 ms) or long (300 ms) voltage steps to –5 or –10 mV, from a holding potential of –80 mV (Fig. 8A). Overall, at concentrations of 2 and 10 nM, ANG II reduced IT-Ca insignificantly to 0.96 ± 0.02 (n = 6) and 0.97 ± 0.01 (n = 3) of its control amplitude (Fig. 8A).

ANG II also failed to alter the amplitude of IT-Ca measured over a wide range of test voltages. In the experiments illustrated in Fig. 8B, I T-Ca was activated by voltage steps between –60 and +50 mV before and after the cell was superfused with ANG II (2 nM) for 7–10 min. Plotting the averaged current densities from three cells against membrane voltage showed that ANG II did not significantly change IT-Ca at any of the 12 test potentials.


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It was discovered that bovine AZG cells express bTREK-1 background K+ channels that set the resting membrane potential and couple ANG II receptor activation to membrane depolarization. These results suggest a model for aldosterone secretion wherein inhibition of bTREK-1 K+ channels by ANG II leads to depolarization and the activation of voltage-gated Ca2+ channels. Accordingly, TREK-1 K+ channel openers reverse ANG II-stimulated depolarization and inhibit aldosterone secretion. The pivotal role assigned to TREK-1 K+ channels in this model contrasts with previous studies that focused on a direct effect of ANG II on T-type Ca2+ channels. In our experiments, ANG II failed to produce a measurable change in the activity of T-type Ca2+ channels under conditions where bTREK-1 currents were nearly completely inhibited by this peptide hormone.

TREK-1 Is the Major Background K+ Channel of Bovine AZG Cells

The combination of Northern blot, in situ hybridization, and patch clamp studies revealed that TREK-1, rather than TASK-3, is the major K+ channel expressed by bovine AZG cells. Northern blot analysis showed that AZF and subcapsular AZG cells both expressed the same three bTREK-1 mRNA transcripts, each of which were similarly induced by forskolin. Thus bTREK-1 expression in AZG cells is likely regulated at the transcriptional level by ACTH through a cAMP-dependent mechanism as it is in AZF cells (14). In situ hybridization on bovine adrenal gland sections corroborated the Northern blot results and showed that bTREK-1 mRNA was uniformly distributed in AZF and AZG cells but was virtually undetectable in the adrenal medulla.

Results from patch clamp experiments were in agreement with those measuring TREK-1 mRNA distribution in the bovine adrenal gland. Bovine AZG cells expressed two K+ currents that were indistinguishable from those of AZF cells. Most importantly, these cells expressed hundreds of background K+ channels that are either dormant of have a low open probability and display a composite profile that matches that of bTREK-1. These outwardly rectifying channels were activated by intracellular acidification, ATP, AA, and CDC and inhibited by ANG II. Of the 2P/4TMS channels identified thus far, only bTREK-1 channels possess all of these properties (15, 16, 53). However, our results do not formally exclude the unlikely possibility that a background K+ channel in addition to bTREK-1 is expressed by bovine AZG cells.

Although we were extremely careful in our dissection to obtain only subcapsular glomerulosa tissue, it is possible that isolated glomerulosa cells were contaminated with a small fraction of AZF cells. By choosing smaller cells, we further reduced the possibility that patch clamp recordings mistakenly included AZF cells. In this regard, it is important to note that each of the more than 25 AZG cells exposed to the bTREK-1 openers responded with large increases in the noninactivating K+ current. Thus bTREK-1 appears to be uniformly expressed in both AZF and AZG cells.

Although TASK-3 may be the major background K+ current found in rat AZG cells (10), we found no evidence that this channel is expressed in bovine AZG. TASK-3 was undetectable in Northern blots of bovine AZG mRNA. When whole cell K+ currents were recorded with pipette solutions that minimized the expression of TREK-1, no background K+ current was activated. There is little doubt that bTREK-1 is the major background channel that sets the resting potential of bovine AZG cells.

In contrast to its expression in the adrenal cortex, no evidence of TREK-1 expression in neural crest-derived chromaffin cells was found in in situ hybridization or whole cell patch clamp experiments. When K+ currents were recorded from chromaffin cells with acidified pipette solution containing 5 mM MgATP, no bTREK-1 current was detected. Furthermore, neither CDC nor AA activated such a current in chromaffin cells (unpublished observations). Although these neural crest-derived cells express multiple K+ channel subtypes, including voltage- and Ca2+-activated K+ channels, the background K+ channel that sets their resting membrane potential remains to be identified (28).

ANG II Regulates AZG Membrane Potential through TREK-1 Inhibition: Model for Depolarization-Dependent Secretion

A requirement for Ca2+ in ANG II-stimulated aldosterone secretion is well established (7, 24, 25, 41, 47). In exploring the cellular mechanism, a number of studies have focused on ANG II modulation of T-type Ca2+ channels (6, 9, 26, 31). However, none of these has provided a satisfactory explanation whereby ANG II could trigger large increases in Ca2+ entry through voltage-gated channels.

The results of the present study suggest a specific model for ANG II-stimulated secretion that assigns a critical role to bTREK-1 K+ channels. In this model, ANG II-mediated inhibition of bTREK-1 is coupled to membrane depolarization, Ca2+ channel activation, and aldosterone secretion. This model allows for the indirect activation of T- or L-type Ca2+ channels through TREK-1 inhibition. Several possibilities exist that could produce efficient Ca2+ entry through either channel. If ANG II-mediated inhibition of bTREK-1 produces a sustained depolarization under physiological conditions, it would produce a continuous influx through noninactivating Ca2+ channels. The effects of ANG II on L-type Ca2+ channels in AZG cells are complex, and both enhancements and inhibition of Ca2+ entry have been reported (2325, 30, 46). However, no L-type Ca2+ current was present in the majority of freshly cultured bovine AZG cells.

Alternatively, bTREK-1 may serve as a brake on the electrical activity of AZG cells. Inhibition of bTREK-1 by ANG II could remove this brake, triggering Ca2+-dependent action potentials driven by opposing T-type Ca2+ currents and A-type K+ currents. Regenerative Ca2+-dependent action potentials in AZG cells have been observed (42). It is unlikely that bovine AZG cells generate action potentials when membrane potential is recorded at 21–23°C with a whole cell patch electrode. Because membrane potential was sampled at 100-ms intervals in these experiments, fast action potentials would not have been readily detected. Action potentials would most likely be recorded from AZG cells in an adrenal slice with a sharp intracellular electrode at physiological temperatures. Regardless, the combined direct effects of ANG II on both T-type Ca2+ channels and bTREK-1 K+ channels produce the ionic effects that mediate aldosterone secretion.

Similar to ANG II, vasopressin stimulates aldosterone secretion through a PLC-coupled receptor requiring Ca2+ influx (21, 44, 50). The inhibition of bTREK-1 K+ channels in AZG cells by vasopressin suggests that it may also stimulate aldosterone secretion through depolarization-dependent Ca2+ entry.

Signaling Pathways for ANG II Modulation of Ion Channels in AZG Cells

The signaling pathways by which ANG II modulates the activity of ion channels in AZG cells are incompletely understood. In particular, the modulation of T-type Ca2+ channels by ANG II has produced conflicting results. In several studies, it has been reported that ANG II increased IT-Ca by a mechanism that involved a hyperpolarizing shift in the voltage dependence of channel activation (6, 9, 27, 31). These effects may occur through Ca2+-dependent activation of Ca2+-CaM kinase II (1, 6, 27).

However, ANG II has also been reported to inhibit T-type Ca2+currents in bovine AZG cells through activation of protein kinase C (45). Notably, the activity of protein kinase C by diacylglycerol is also enhanced by [Ca2+]i (37). Finally, in perforated patch whole cell recordings, ANG II failed to alter the activity of IT-Ca in rat AZG cells (25).

Although our results do not explain the conflicting findings described above, they do suggest that ANG II modulates ion channels in AZG cells by multiple signaling pathways, not all of which are Ca2+ dependent. In our experiments, including those measuring the activity of bTREK-1 and T-type Ca2+ channels, [Ca2+]i was strongly buffered to 22 nM using 11 mM BAPTA. If ANG II modulation of IT-Ca, including activation or inhibition, requires activation of Ca2+-dependent enzymes, this response would likely be blunted or eliminated in our experiments (2, 54).

Regardless, our results do show that ANG II potently and nearly completely inhibits bTREK-1 current and depolarizes AZG cells through a signaling pathway that does not modulate T-type Ca2+ channels. It appears that ANG II-mediated modulation of Ca2+ and K+ channels in bovine AZG cells occurs through multiple Ca2+-dependent and -independent signaling pathways.

TREK-1 Channel Activation and Inhibition of Aldosterone Secretion

The ability of AA and CDC to activate bTREK-1 channels and restore membrane potential to AZG cells depolarized by ANG II provides convincing evidence that these channels set the resting potential of AZG cells. The effectiveness of CDC in reversing ANG II-stimulated depolarization and inhibiting ANG II-stimulated aldosterone secretion is consistent with a model in which TREK-1 couples ANG II receptor activation to depolarization-dependent Ca2+ entry. Within this framework, CDC would negate the membrane depolarization that leads to Ca2+ channel activation.

These results also illustrate the utility of this new type of K+ channel activator in exploring the function of 2P/4TMS channels in cell physiology. However, additional studies will be needed to determine its specificity as an ion channel modulator. AA and other cis polyunsaturated fatty acids that activate the same subgroup of 2P/4TMS background channels also modulate several types of voltage-gated channels, limiting their value as pharmacological tools (38, 49).

In summary, the results of the present and previous studies clearly demonstrate that bTREK-1 is the predominant background K+ channel that sets the resting membrane potential of both bovine AZF and AZG cells and couples ACTH and ANG II receptors to depolarization-dependent Ca2+ entry and the secretion of cortisol and aldosterone. In contrast, TASK-type K+ channels may function similarly in the rat adrenal cortex, at least in the adrenal glomerulosa (10, 11).

Thus significant species differences that exist between rat and bovine adrenal cortical background K+ channels highlight the importance of identifying the background K+ channels in the human adrenal cortex. PCR using human cDNA as the template showed that TREK-1 is strongly expressed in the human adrenal (data not shown). Additional experiments will be required to determine whether TREK-1 functions in the human adrenal cortex as it does in bovine AZF and AZG. If so, then TREK-1 channel activators might serve as therapeutic agents in endocrine diseases marked by excessive cortisol or aldosterone secretion.


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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-47875 (to J. J. Enyeart), and in part by the National Science Foundation under Agreement 0112050 (S. Danthi).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Enyeart, Dept. of Neuroscience, The Ohio State Univ. College of Medicine and Public Health, 5196 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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Barrett PQ, Lu HK, Colbran R, Czerik A, and Pancrazio JJ. Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II. Am J Physiol Cell Physiol 279: C1694–C1703, 2000.[Abstract/Free Full Text]
  2. Beech DJ, Berheim L, Mathie A, and Hille B. Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons. Proc Natl Acad Sci USA 88: 652–656, 1991.[Abstract/Free Full Text]
  3. Bondy PK. Diseases of the Adrenal Gland. In: Williams Textbook of Endocrinology, Philadelphia, PA: Saunders, 1985, p. 816–890.
  4. Brauneis U, Vassilev PM, Quinn SJ, Williams GH, and Tillotson DL. ANG II blocks potassium currents in zona glomerulosa cells from rat, bovine and human adrenals. Am J Physiol Endocrinol Metab 260: E772–E779, 1991.[Abstract/Free Full Text]
  5. Brooks SPJ and Storey KB. Bound and determined: a computer program for making buffers of defined ion concentrations. Anal Biochem 201: 119–126, 1992.[CrossRef][ISI][Medline]
  6. Chen XL, Bayliss DA, Fern RJ, and Barrett PQ. A role for T-type Ca2+ channels in the synergistic control of aldosterone production by ANG II and K+. Am J Physiol Renal Physiol 276: F674–F683, 1999.[Abstract/Free Full Text]
  7. Cherradi N, Brandenburger Y, Rossier MF, Vallotton MB, Stocco DM, and Capponi AM. Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells. Mol Endocrinol 12: 962–972, 1998.[Abstract/Free Full Text]
  8. Cho H, Ueda M, Tamaoka M, Hamaguchi M, Aisaka K, Kiso Y, Inoue T, Ogino R, Tatsuoka T, and Ishihara T. Novel caffeic acid derivatives: extremely potent inhibitors of 12-lipoxygenase. J Med Chem 34: 1503–1505, 1991.[CrossRef][ISI][Medline]
  9. Cohen CJ, McCarthy RT, Barrett PQ, and Rasmussen H. Ca channels in adrenal glomerulosa cells: K+ and angiotensin II increase T-type Ca channel current. Proc Natl Acad Sci USA 85: 2412–2416, 1988.[Abstract/Free Full Text]
  10. Czirjak G and Enyedi P. TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells. Mol Endocrinol 16: 621–629, 2002.[Abstract/Free Full Text]
  11. Czirjak G, Fischer T, Spat A, Lesage F, and Enyedi P. TASK (TWIK-related acid-sensitive K+ channel) is expressed in glomerulosa cells of rat adrenal cortex and inhibited by angiotensin II. Mol Endocrinol 14: 863–874, 2000.[Abstract/Free Full Text]
  12. Danthi S, Enyeart JA, and Enyeart JJ. Modulation of native TREK-1 and Kv1.4 channels by polyunsaturated fatty acids and lysophospholipids. J Membr Biol 195: 147–164, 2003.[CrossRef][ISI][Medline]
  13. Danthi S, Enyeart JA, and Enyeart JJ. Caffeic acid esters activate TREK-1 potassium channels and inhibit depolarization-dependent secretion. Mol Pharmacol 65: 1–12, 2004.[Abstract/Free Full Text]
  14. Enyeart JA, Danthi SJ, and Enyeart JJ. Corticotropin induces the expression of TREK-1 mRNA and K+ current in adrenocortical cells. Mol Pharmacol 64: 132–142, 2003.[Abstract/Free Full Text]
  15. Enyeart JJ, Gomora JC, Xu L, and Enyeart JA. Adenosine triphosphate activates a noninactivating K+ current in adrenal cortical cells through nonhydrolytic binding. J Gen Physiol 110: 679–692, 1997.[Abstract/Free Full Text]
  16. Enyeart JJ, Xu L, Danthi S, and Enyeart JA. An ACTH- and ATP-regulated background K+ channel in adrenocortical cells is TREK-1. J Biol Chem 277: 49186–49199, 2002.[Abstract/Free Full Text]
  17. Enyeart JA, Xu L, and Enyeart JJ. A bovine adrenocortical Kv1.4 K+ channel whose expression is potently inhibited by ACTH. J Biol Chem 275: 34640–34649, 2000.[Abstract/Free Full Text]
  18. Fink M, Lesage F, Duprat F, Heurteaux C, Reyes R, Fosset M, and Lazdunski M. A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids. EMBO J 17: 3297–3308, 1998.[Abstract/Free Full Text]
  19. Goldstein SA, Bockenhauer D, O'Kelly I, and Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2: 175–184, 2001.[CrossRef][ISI][Medline]
  20. Gu J, Wen Y, Mison A, and Nadler JL. 12-Lipoxygenase pathway increases aldosterone production, 3',5'-cyclic adenosine monophosphate response element-binding protein phosphorylation, and p38 mitogen-activated protein kinase activation in H295R human adrenocortical cells. Endocrinology 144: 534–543, 2003.[Abstract/Free Full Text]
  21. Guillon G, Balestre MN, Chouinard L, and Gallo-Payet N. Involvement of distinct G proteins in the action of vasopressin on rat glomerulosa cells. Endocrinology 126: 1699–1708, 1990.[Abstract]
  22. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[CrossRef][ISI][Medline]
  23. Hunyady L, Rohacs T, Bago A, Deak F, and Spat A. Dihydropyridine-sensitive initial component of the ANG II-induced Ca2+ response in rat adrenal glomerulosa cells. Am J Physiol Cell Physiol 266: C67–C72, 1994.[Abstract/Free Full Text]
  24. Kojima I, Kojima K, and Rasmussen H. Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. J Biol Chem 260: 9171–9176, 1985.[Abstract/Free Full Text]
  25. Lotshaw DP. Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion. Mol Cell Endocrinol 175: 157–171, 2001.[CrossRef][ISI][Medline]
  26. Lu HK, Fern RJ, Luthin D, Linden J, Liu LP, Cohen CJ, and Barrett PQ. Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, Gi. Am J Physiol Cell Physiol 271: C1340–C1349, 1996.[Abstract/Free Full Text]
  27. Lu HK, Fern RJ, Nee JJ, and Barrett PQ. Ca2+-dependent activation of T-type Ca2+ channels by calmodulin-dependent protein kinase II. Am J Physiol Renal Fluid Electrolyte Physiol 267: F183–F189, 1994.[Abstract/Free Full Text]
  28. Marty A and Neher E. Potassium channels in cultured bovine adrenal chromaffin cells. J Physiol 367: 117–141, 1985.[Abstract]
  29. Matsunaga H, Maruyama Y, Kojima I, and Hoshi T. Transient Ca2+-channel current characterized by a low-threshold voltage in zona glomerulosa cells of rat adrenal cortex. Pflügers Arch 408: 351–355, 1987.[CrossRef][ISI][Medline]
  30. Maturana AD, Casal AJ, Demaurex N, Vallotton MB, Capponi AM, and Rossier MF. Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells. J Biol Chem 274: 19943–19948, 1999.[Abstract/Free Full Text]
  31. McCarthy RT, Isales C, and Rasmussen H. T-type calcium channels in adrenal glomerulosa cells: GTP-dependent modulation by angiotensin II. Proc Natl Acad Sci USA 90: 3260–3264, 1993.[Abstract/Free Full Text]
  32. Mlinar B, Biagi BA, and Enyeart JJ. A novel K+ current inhibited by ACTH and angiotensin II in adrenal cortical cells. J Biol Chem 268: 8640–8644, 1993.[Abstract/Free Full Text]
  33. Mlinar B, Biagi BA, and Enyeart JJ. Losartan-sensitive AII receptors linked to depolarization-dependent cortisol secretion through a novel signaling pathway. J Biol Chem 270: 20942–20951, 1995.[Abstract/Free Full Text]
  34. Mlinar B and Enyeart JJ. Voltage-gated transient currents in bovine adrenal fasciculata cells II: A-type K+ current. J Gen Physiol 102: 239–255, 1993.[Abstract]
  35. Morohashi K, Yoshioka H, Gotoh O, Okada Y, Yamamoto K, Miyata T, Sogawa K, Fujii-Kuriyama Y, and Omura T. Molecular cloning and nucleotide sequence of DNA of mitochondrial cytochrome P-450(11 beta) of bovine adrenal cortex. J Biochem 102: 559–568, 1987.[Abstract]
  36. Nadler JL, Natarajan R, and Stern N. Specific action of the lipoxygenase pathway in mediating angiotensin II-induced aldosterone synthesis in isolated adrenal glomerulosa cells. J Clin Invest 80: 1763–1769, 1987.[ISI][Medline]
  37. Oancea E and Meyer T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95: 307–318, 1998.[CrossRef][ISI][Medline]
  38. Ordway RW, Singer JJ, and Walsh JVJ. Direct regulation of ion channels by fatty acids. Trends Neurosci 14: 96–100, 1991.[CrossRef][ISI][Medline]
  39. Patel AJ and Honore E. Properties and modulation of mammalian 2P domain K+ channels. Trends Neurosci 24: 339–346, 2001.[CrossRef][ISI][Medline]
  40. Python CP, Rossier MF, Valloton MB, and Capponi AM. Peripheral-type benzodiazepines inhibit calcium channels and aldosterone production in adrenal glomerulosa cells. Endocrinology 132: 1489–1496, 1993.[Abstract]
  41. Quinn SJ. Regulation of aldosterone secretion. Ann Rev Physiol 50: 409–426, 1988.[CrossRef][ISI][Medline]
  42. Quinn SJ, Cornwall MC, and Williams GH. Electrical properties of isolated rat adrenal glomerulosa and fasciculata cells. Endocrinology 120: 903–914, 1987.[Abstract]
  43. Quinn SJ, Cornwall MC, and Williams GH. Electrophysiological responses to Angiotensin II of isolated rat adrenal glomerulosa cells. Endocrinology 120: 1581–1589, 1987.[Abstract]
  44. Quinn SJ, Enyedi P, Tillotson DL, and Williams GH. Kinetics of cytosolic calcium and aldosterone responses in rat adrenal glomerulosa cells. Endocrinology 129: 2431–2441, 1991.[Abstract]
  45. Rossier MF, Aptel HB, Python CP, Burnay MM, Valloton MB, and Capponi AM. Inhibition of low threshold calcium channels by angiotensin II in adrenal glomerulosa cells through activation of protein kinase C. J Biol Chem 270: 15137–15142, 1995.[Abstract/Free Full Text]
  46. Rossier MF, Burnay MM, Vallotton MB, and Capponi AM. Distinct functions of T- and L-type calcium channels during activation of bovine adrenal glomerulosa cells. Endocrinology 137: 4817–4826, 1996.[Abstract]
  47. Rossier MF, Ertel EA, Vallotton MB, and Capponi AM. Inhibitory action of mibefradil on calcium signaling and aldosterone synthesis in bovine adrenal glomerulosa cells. J Pharmacol Exp Ther 287: 824–831, 1998.[Abstract/Free Full Text]
  48. Schneider EG. Effect of vasopressin on adrenal steroidogenesis. Am J Physiol Regul Integr Comp Physiol 255: R806–R811, 1988.[Abstract/Free Full Text]
  49. Vgeugdenhil M, Bruehl C, Voskuyl RA, Kang JX, Leaf A, and Wadman WJ. Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons. Neurology 93: 12559–12563, 1996.
  50. Woodcock EA, McLeod JK, and Johnston CI. Vasopressin stimulates phosphatidylinositol turnover and aldosterone synthesis in rat adrenal glomerulosa cells: comparison with angiotensin II. Endocrinology 118: 2432–2436, 1986.[Abstract]
  51. Xu L and Enyeart JJ. Adenosine inhibits a noninactivating K+ current in adrenal cortical cells by activation of multiple P1 receptors. J Physiol (Camb) 521.1: 81–97, 1999.
  52. Xu L and Enyeart JJ. Purine and pyrimidine nucleotides inhibit a non-inactivating K+ current and depolarize adrenal cortical cells through a G protein-coupled receptor. Mol Pharmacol 55: 364–376, 1999.[Abstract/Free Full Text]
  53. Xu L and Enyeart JJ. Properties of ATP-dependent K+ channels in adrenocortical cells. Am J Physiol Cell Physiol 280: C199–C215, 2001.[Abstract/Free Full Text]
  54. Yu SP, O'Malley DM, and Adams PR. Regulation of M current by intracellular calcium in bullfrog sympathetic ganglion neurons. J Neurosci 14: 3487–3499, 1994.[Abstract]