A novel K+ current inhibited by adrenocorticotropic hormone and angiotensin II in adrenal cortical cells.

Adrenocorticotropic hormone (ACTH) and angiotensin II (AII) are peptides that regulate the production of steroid hormones by cells of the adrenal cortex. The cellular mechanisms linking these peptides to corticosteroid hormone secretion are not understood. In patch clamp recordings from bovine adrenal zona fasciculata (AZF) cells, we have identified a novel cholera toxin-sensitive K+ current (IAC), which is potently inhibited by both ACTH and AII with respective EC50 values of 4.5 and 145 pM. These two peptides depolarize AZF cells with a temporal pattern and potency that parallels the inhibition of IAC. With the discovery of IAC, we have identified a common molecular target for both ACTH and AII. The convergent inhibition of IAC by these two peptides suggests a mechanism whereby biochemical signals originating at the cell membrane can be transduced to depolarization-dependent Ca2+ entry and steroid hormone secretion.

Transmitter and hormone release by many secretory cells is tightly coupled to depolarization-dependent Ca2+ entry. A requirement for Ca2+ in steroidogenesis by cells of the adrenal cortex is well established (1,2). Several lines of evidence indicate that ACTH' and AII-stimulated cortisol and aldosterone production involve Ca2+ entry through voltage-gated channels (3-8). Although separate signaling pathways and second messengers for these regulatory peptides have been described (9)(10)(11), no specific mechanism linking the peptide receptors to membrane depolarization and corticosteroid secretion has been discovered. In particular, ion channels common to steroid secreting cells that control membrane potential and whose modulation by peptides would allow depolarization-dependent Ca2+ entry and secretion have not been identified. In this report, we describe a novel K+ current IAc, which is co-expressed with an A-type K+ current in bovine AZF cells. IAc displays properties expected of a K+ channel that * This work was supported by National Institute for Diabetes and Digestive and Kidney Disorders Grant DK-40131 (to J. J. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be ad- toxin, pertussis toxin, tetraethylammonium, 4-aminopyridine, and apamin were obtained from Sigma. a-Dendrotoxin was obtained from Alomone Laboratories (Jerusalem, Israel).
Isolation and Culture of AZF Cells-Bovine adrenal glands were obtained from steers (age range 1-3 years) 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 phosphatebuffered saline containing 0.2% dextrose. Isolated AZF cells were prepared as previously described (12) with some modifications. In a sterile tissue culture hood, the adrenals were cut in half lengthwise and the lighter medulla tissue trimmed away from the cortex and discarded. The capsule with attached glomerulosa and thicker fasciculata-reticularis layer was then dissected into pieces approximately 1.0 X 1.0 X 0.5 cm. A Stadie-Riggs tissue slicer (Thomas Scientific) was used to slice fasciculata-reticularis tissue from the glomerulosa layers. Fasciculata-reticularis slices were diced into 0.5-mm3 pieces and dissociated with 2 mg/ml (about 200 units/ml) of Type I collagenase, 0.2 mg/ml deoxyribonuclease in MEM plus 100 units/ml penicillin, 0.1 mg/ml streptomycin for approximately 45 min at 37 "C in a shaking water bath, triturating after 15 and 30 min with a sterile, plastic transfer pipette. After incubating, the suspension was filtered through one layer of sterile cheesecloth, centrifuged to pellet cells at 100 X g for 5 min. The cells were then washed twice with MEM, centrifuging as before to pellet. Cells were filtered through 200-pm stainless steel mesh to remove clumps after resuspending in MEM. Dispersed cells were again centrifuged and either resuspended in DMEM/Ham's F-12 (1:l) with 10% FBS, 100 units/ml penicillin, 0.1 mg/ml streptomycin and plated for immediate use or resuspended in FBS,, 5% MezSO, divided into 1-ml aliquots each containing about 1 X IO7 cells, and stored in liquid nitrogen for future use. Cells were plated in 35-mm dishes containing 9-mm2 glass coverslips, which had been treated with fibronectin (10 pg/ml) at 37 "C for 30 min and then rinsed twice with warm, sterile phosphate-buffered saline immediately before adding cells. Dishes were maintained at 37 "C in a humidified atmosphere of 95% air and 5% COZ.
The functional state of isolated AZF cells was determined by measuring cortisol secretion from cultured cells under basal conditions and in response to the pituitary hormone ACTH. Enzymatically plates at a density of about 4 x 10' cells/dish in DMEM/F-12 (1:1) dissociated AZF cells were cultured on fibronectin-treated 35-mm as described above. After 24 h, media was replaced with control media or the same media containing lo-' ACTH . Cells were returned to the incubator for 24 h at which time media was collected and cortisol measured using a solid phase radioimmunoassay kit solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl, 2 mM M&lz, 10 mM HEPES, and 5 mM glucose, pH 7.4, using NaOH. Deviations from these solutions are noted in the text. All solutions were filtered through 0.22-pm cellulose acetate filters.
AZF cells were used for patch clamp experiments 2-48 h after plating. Typically, cells with diameters of 15-30 pm and capacitances of 15-35 picofarads 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 4-6 ml/min. Patch electrodes with resistances of 1.0-2.0 megohms were fabricated from Corning RC-6 or 0010 glass (Garner Glass Co., Claremont, CAI. K+ currents were recorded at room temperature (22-24 "C) following the procedure of Hamill et al. (13) using a List EPC-7 patch clamp amplifier.
Pulse generation and data acquisition were done using an IBM-AT computer and PCLAMP software with an Axolab interface (Axon Instruments, Inc., Burlingame, CA). Currents were digitized at 1-50 kHz after filtering with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using scaled hyperpolarizing steps of 1/2 to 1/4 amplitude. Data were analyzed and plotted using PCLAMP (CLAM-PAN and CLAMPFIT) and GraphPAD InPLOT. Drugs were applied by bath perfusion, controlled manually by a six-way rotary valve.

RESULTS
In whole-cell patch clamp recordings, two distinct components of K+ current were expressed in nearly all of more than 200 AZF cells studied. These included a rapidly inactivating A-type component, described in detail elsewhere,' and a novel non-inactivating current, which in view of its location in cells of the adrenal cortex, we have named IAC. During prolonged recordings, IAC amplitude increased dramatically from 3.73 k 0.8 pA/picofarads ( n = 45) to 34.98 k 5.2 pA/picofarads ( n = 54) approaching the apparent maximum value with a halftime of 8.5 k 0.5 min (n = 28). The amplitude of the transient K+ current typically remained nearly constant in these same recordings (Fig. hi). The time-dependent growth of macroscopic IAC was accompanied by a large increase in current noise indicative of a relatively large unitary conductance.
In addition to widely differing inactivation kinetics, IAC was clearly distinguished from A-type current by its voltage-independent availability. At a holding potential of -40 mV, the transient K+ current was completely inactivated while IAC was unaffected. When IAC was viewed in isolation under these conditions, no delay in current onset was apparent with depolarizing steps to potentials up to +10 mV, suggesting that IAC channels remain open at the holding potential (Fig. 1B).
Current-voltage relationships obtained from several cells that expressed negligible A-type current indicated that IAC channels were open at the resting membrane potential of these cells (-71.1 mV) (Fig. 1C). In contrast, the threshold for A current activation was approximately -40 mV. Although blocked by several K+ channel antagonists, IAC was pharmacologically distinguishable from the A-type K+ current in AZF cells. At a concentration of 2 mM, 4-aminopyridine inhibited the A-type current activated by voltage steps to +20 mV almost completely, but reduced I AC by only 19.6 f 9.5% (n = 6). A second K+ channel blocker, tetraethylammonium (20 mM), inhibited IAC by 57.3 k 4.1% ( n = 3). IAC was insensitive to wdendrotoxin and apamin at concentrations up to 500 nM, but disappeared completely when CsCl replaced KC1 in the recording electrode.
The growth of IAC in whole cell recordings suggested that B. Mlinar and J. J. Enyeart, submitted for publication. A, timedependent growth of IAC. K+ currents were activated at 30-5 intervals by voltage steps to +20 mV from a holding potential of -80 mV. Current records at indicated times after initiating whole-cell recordings. B and C, current-voltage relationships for IAc. B, current records in response to voltage steps of various sizes applied at 30-5 intervals from a holding potential of -40 mV. C, IAC was measured in a cell with no detectable inactivating K+ current at potentials ranging from -80 to +40 mV. Test pulse was a 2-5 ramp applied from a holding potential of 0 mV beginning at -80 mV. Top trace is current plotted against test potential corrected for series resistance error, in control saline. Bottom truce is current from same cell after superfusion of 10 PM ACTH. this current was under the inhibitory control of an intracellular factor affected by cell dialysis. The time-dependent increase in IAc was completely inhibited by including the nonhydrolyzable G T P analog GTPyS (150-350 p~) in the recording pipette (Fig. 2, A and B ) . The inhibitory effect of GTPrS indicated modulation of IAC by a GTP-binding protein. We further tested this possibility by preincubating cells with bacterial toxins from Vibrio cholera (CTx) and Bordatella pertussis (PTx), which, respectively, activate Gs and suppress activation of Gi and Go. Functional expression of IAC was affected only by CTx, which completely inhibited its appearance, indicating that IAC was under the inhibitory control of Gs (Fig. 2, A and B ) . The expression of A-type K+ current was not changed by guanine nucleotides or bacterial toxins.
T o determine whether I AC might be modulated by peptides that physiologically regulate cortisol production, AZF cells were exposed to ACTH and AI1 while recording K+ currents.
IAc was potently and selectively inhibited by both peptides. ACTH produced complete inhibition of IAC with an ECm of 4.1 PM (Fig. 3, A and C). The current was inhibited equally at potentials from -80 to +40 mV (Fig. IC). AI1 was also effective at subnanomolar concentrations (EC, = 145 pM), but IAc was reduced by a maximum of 77.5 f 2.8% (Fig. 3C). The inhibition of IAc by both peptides began after a delay of 1-2 min, and typically, several additional minutes were required before maximum inhibition was achieved (Fig. 3, A  and B ) . When the inactive guanine nucleotide GDPpS (0.5-1 p~) replaced GTP in the pipette, inhibition of I AC by both peptides was blunted. Under these conditions, 500 PM ACTH reduced IAc by 84.0 -C 6.4% ( n = 4), while 10 nM AI1 inhibited the current by 56.8 k 17.3% (n = 4) (data not shown).
In single channel recordings from outside-out patches, a non-inactivating ACTH-sensitive K+ channel was identified.
Recordings from a membrane patch containing two such channels and corresponding amplitude histogram are shown in Fig. 4 (A and C). In control saline, channel activity was marked by long openings lasting up to hundreds of milliseconds. Two min after superfusing the patch with 1 nM ACTH, channel opening had ceased (Fig. 4, B and D). The currentvoltage relationship indicated that the ACTH-sensitive channel was outwardly rectifying with a major conductance state of 60-65 picosiemens at potentials positive to +20 mV (Fig.  4E). A unitary conductance of this size clearly distinguishes this channel from known A-type K+ channels.  (Fig.  5A). AI1 depolarized cells by a similar amount (52.6 f 0.9 mV), but this peptide was again less potent (EC, = 283 p M ) (Fig. 5A). Onset of depolarization by either peptide occurred after a delay of one to several minutes and was reversed upon superfusion with saline (Fig. 5, B and C).

DISCUSSION
The discovery of a novel K+ channel that sets AZF cell membrane potential while it is potently inhibited by both ACTH and AI1 suggests a specific mechanism for peptide hormone-stimulated corticosteroid secretion, which emphasizes the importance of electrical events and Ca2+ entry. In addition to the two types of K+ current described above, all points amplitude histograms constructed from 48 consecutive 400-ma 0 sweeps (97,152 total points), before ( A ) and 5 min after ( B ) exposing the patch to 1 nM ACTH. Cutoff frequency is 1 kHz. C and D, representative 400-ma sweeps before ( C ) and after (D) ACTH.
Scale bars, 2 pA and 20 ms. E, singlechannel IV. Single-channel conductance was obtained for a patch containing one channel at various test potentials from FIG. 5. Depolarization of AZF cells by ACTH and AII. Concentration dependence and temporal pattern are shown. After impaling a cell and obtaining a stable resting potential, cells were superfused with ACTH  or AI1 at various concentrations while continuously recording membrane potential. A , concentration dependence; maximum depolarization is plotted against peptide concentration. Values are mean & S.E. of 5-9 separate determinations. B and C, time course and reversal of depolarization by 50 p M ACTH and 250 p~ AII. Cells were superfused with peptides and control solution at the indicated times.
bovine AZF cells express a low voltage-activated T-type Ca2+ current (15). We propose that these channels determine the electrical properties of AZF cells and transduce biochemical signals originating at the cell membrane to depolarizationdependent Ca2+ entry and secretion. Specifically, ACTH and AI1 acting through separate receptors inhibit IAC, triggering the sequence of membrane depolarization, Ca2+ channel activation, and corticosteroid production. In this regard, we have found that T-type Ca2+ channel antagonists block ACTHand AII-stimulated cortisol production and Ca2+ current a t similar concentrations (15 been reported to modulate both Ca2+ and transient K+ currents in rat and bovine adrenal glomerulosa cells (3,5,17,18), these peptides did not affect T3 or A currents in bovine AZF cells. ACTH and AI1 are known to act on adrenal cortical cells through separate signaling pathways. ACTH stimulates the synthesis of CAMP, whereas AI1 enhances the production of inositol 1,4,5-trisphosphate and diacylglycerol through activation of phospholipase C. Whether the convergent inhibition of IAc by these two peptides indicates a similar convergence of different second messengers is not known. ACTH blocked IAc and depolarized AZF cells in our study at low picomolar concentrations that produce no measurable increase in cAMP (19,20). Thus it appears unlikely that either of these effects is mediated by this second messenger. In this regard, previous studies on adrenal cells have led to the conclusion that cAMP is the primary intracellular mediator of ACTH-stimulated steroidogenesis (21)(22)(23). This notion remains even though ACTH triggers Ca2+ uptake and stimulates corticosteroid production at concentrations far lower than those required to produce increases in cAMP (8, 10,19,24). The excellent correlation between ACTH-stimulated inhibition of IAC and membrane depolarization with Ca2+ influx and corticosteroid secretion suggest that Ca2+ may be the primary intracellular messenger, particularly a t low ACTH concentrations.
Activation of phospholipase C by AI1 enhances the synthesis of inositol 1,4,5-trisphosphate and diacylglycerol and triggers the release of Ca2+ from intracellular stores. Whether one of these second messengers inhibits I AC is not known.
Although Ca2+-mediated inhibition of K+ channels has been observed in several cell types (14,25,26), it is unlikely that Ca2+ released from intracellular stores inhibited I AC in our experiments since intracellular Ca2+ was highly buffered by BAPTA.
Our results demonstrating inhibition of I AC by GTP+ and CTx indicate that the corresponding channels are under the inhibitory control of a Gs protein. This may be the first outwardly rectifying K+ channel to be inhibited by this GTPbinding protein. An outwardly rectifying K+ current, which is present at negative potentisls and modulated by G-protein activation, has been identified in a leukemic cell line (16). In spite of some resemblance to IAC, the current in leukemic cells differs in several important respects. In particular, the func-tional expression of the leukemic cell Kf current is blocked by PTx but not CTx indicating the inhibitory control of Gi or Go rather than G.. The small unitary conductance of the leukemic cell K+ channel (8 picosiemens) and its activation by GTPyS also clearly distinguish it from IAc.
It is still unclear whether Gs links the activation of ACTH and AI1 receptors to inhibition of IAC. In this regard, channel modulation that occurs through a G-protein intermediate usually occurs either by a membrane-delimited tight receptor coupling or through a diffusible second messenger. The responses we observed with the two peptides are not consistent with either of these mechanisms. The delay of several minutes required for ACTH or AI1 to inhibit IAc or to depolarize AZF cells is quite long even for responses requiring synthesis of intracellular second messengers. Furthermore, both peptides inhibit IAC maximally in cells that have been patch-clamped in the whole-cell mode for periods of 1 h. Responses requiring the synthesis of diffusible second messengers would likely have been "washed out" long before this time.
Regardless of the signal transduction pathways involved, the convergent inhibition of IAc by ACTH and AI1 may represent the major components of a physiological mechanism regulating corticosteroid secretion. As in other secretory cells, the physiological regulation of corticosteroid production by AZF cells appears tightly coupled to the function and modulation of ion channels.