A Stadie-Riggs tissue slicer (Thomas Scientific) was used to slice fasciculata-reticularis tissue ... 100 X g for 5 min. The cells were then washed twice with MEM,.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268, No. 12, Issue of April 25, pp. 864O-?j44, 1993 Printed m U.S. A.
A Novel K+ Current Inhibitedby Adrenocorticotropic Hormone and Angiotensin I1 in Adrenal Cortical Cells* (Received for publication, December 17, 1992,and in revised form, February 9, 1993)
Boris Mlinar, Bruce A. BiagiS, and John J. Enyeart$ From the Department of Pharmacology and Neuroscience Program and the $Department of Physiology, OhioState University, College of Medicine, Columbus, Ohio43210-1239
Adrenocorticotropichormone (ACTH)and angiotensin I1 (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 toxinsensitive K+ current (IAc),which is potently inhibited by both ACTH and AI1 with respective ECao values of 4.5 and 145 p ~ These . two peptides depolarize AZF cells witha temporalpattern and potency that parallels the inhibition of IAC.With the discovery of IAC, we have identified a common molecular target for both ACTH and AIL The convergent inhibition of IACby these two peptides suggests a mechanism whereby biochemical signals originatingat the cell membrane can be transduced to depolarization-dependent Ca2+entry and steroid hormone secretion.
could set themembrane potential of adrenal cells. In addition, it is potently inhibited by both ACTH and AII. MATERIALS AND METHODS
Tissue culture media, antibiotics, fibronectin, and fetal calf serum were obtained from GIBCO. Culture dishes were purchased from Corning (Corning, NY). Coverslips were from Bellco (New Jersey). Enzymes, ACTH(1-24), AII, GTP, MgATP, GDPPS, GTP-yS,cholera 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) Transmitter and hormone release by many secretory cells was used to slice fasciculata-reticularis tissue from the glomerulosa is tightly coupled to depolarization-dependent Ca2+entry. A layers. Fasciculata-reticularis slices were diced into 0.5-mm3 pieces and dissociated with 2 mg/ml (about 200 units/ml) of Type I collarequirement for Ca2+in steroidogenesis by cells of the adrenal genase, 0.2 mg/ml deoxyribonuclease in MEM plus 100 units/ml cortex is well established (1, 2). Several lines of evidence penicillin, 0.1 mg/ml streptomycin for approximately 45 min a t 37 “C indicate that ACTH’ and AII-stimulated cortisol and aldos- in a shakingwater bath, triturating after15 and 30 min with a sterile, terone production involve Ca2+entry through voltage-gated plastic transfer pipette. After incubating, the suspension was filtered channels (3-8). Although separate signaling pathways and through one layer of sterile cheesecloth, centrifuged to pellet cells at second messengers for these regulatory peptides have been 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 described (9-11), no specific mechanism linking the peptide stainless steel mesh to remove clumps after resuspending in MEM. receptors to membrane depolarization and corticosteroid se- Dispersed cells were again centrifuged and either resuspended in cretion has been discovered. In particular, ion channels com- DMEM/Ham’s F-12 (1:l)with 10% FBS, 100 units/ml penicillin, 0.1 mon to steroid secreting cells that control membrane potential mg/ml streptomycin and plated for immediate use or resuspended in and whose modulation by peptides would allow depolariza- FBS,, 5% MezSO, divided into 1-ml aliquots each containing about 1 tion-dependent Ca2+entry and secretion have not been iden- X IO7 cells, and stored in liquid nitrogen for future use. Cells were plated in 35-mm dishes containing 9-mm2glass coverslips, which had tified. In thisreport, we describe a novel K+ current IAc, which been treated with fibronectin (10 pg/ml) at 37 “C for 30 min and then is co-expressed with an A-type K+ current in bovine AZF rinsed twice with warm, sterile phosphate-buffered saline immedicells. IAc displays properties expected of a K+ channel that ately before adding cells. Dishes were maintained at 37 “C ina humidified atmosphere of 95% air and 5%COZ. The functional state of isolated AZF cells was determined by * This work was supported by National Institute for Diabetes and Digestive and Kidney Disorders Grant DK-40131 (to J. J. E.). The measuring cortisol secretion from cultured cells under basal condicosts of publication of this article were defrayed in part by the tions and in response to thepituitary hormone ACTH. Enzymatically payment of page charges. This articlemust therefore be hereby dissociated AZF cells were cultured on fibronectin-treated 35-mm marked “aduertisement” in accordance with 18 U.S.C. Section 1734 plates a t a density of about 4 x 10‘ cells/dish in DMEM/F-12 (1:1) as described above. After24 h, media was replaced with control media solely to indicate this fact. To whom correspondence and reprint requests should be ad- or the same media containing lo-’ ACTH(1-24). Cells were returned dressed Dept. of Pharmacology, Ohio State University College of to the incubator for 24 h a t which time media was collected and Medicine, 5188 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210- cortisol measured using a solid phase radioimmunoassay kit (Diagnostic Products Corp. Los Angeles,CA). Control cells produced 1239. Tel.: 614-292-3511;Fax: 614-292-9805. The abbreviations used are: ACTH, adrenocorticotropic hormone; cortisol at a rate of0.87 +. 0.02 ng/lO‘ cells/24 h. ACTH increased AII, angiotensin 11; AZF, adrenal zona fasciculata; GDPPS, guanyl- cortisol production 93-fold to 80.90 4.26 ng/lO‘ cells/24 h. Solutions and Recording Conditions-Patch clamp recordings of 5’-0-(2-thiodiphosphate);GTPrS, guanosine 5’-0-(3-thiotriphosphate); MEM,minimum Eagle’s medium; DMEM, Dulbecco’s K+ channel currents were made in the whole-cell and outside-out modified Eagle’s medium; BAPTA, 1,2-bis(2-aminophenoxy)ethane- configurations. The standardpipette solution was 120 mM KC1,2 mM N,N,N’,N‘-tetraacetic acid; CTx, bacterial toxin from V. cholera; MgClZ,1 mM CaCI2, 10 mM HEPES, 11 mM BAPTA, 200 PM GTP, 2 mM MgATP with pH buffered to 7.2 using KOH. The external PTx, bacterial toxin from B. pertussis.
*
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8641
ACTH and AII Inhibit a Novel K" Current inAdrenal Cells solution consisted of140 mM NaCl, 5 mM KCl, 2 mM CaCl, 2 mM M&lz, 10 mM HEPES, and 5 mM glucose,pH 7.4, usingNaOH. Deviations from these solutions are noted in the text. All solutions were filtered through0.22-pm celluloseacetate filters. AZFcellswereusedfor 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 culturedishes to the recordingchamber(volume,1.5 ml), which was continuously perfusedby gravity at a rate of 4-6 ml/min. Patch electrodes with resistancesof 1.0-2.0 megohms werefabricated 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 IBMAT computerand 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 stepsof 1/2 to 1/4 amplitude. Datawere analyzed and plotted using PCLAMP (CLAMPAN and CLAMPFIT) and GraphPAD InPLOT. Drugs were applied by bath perfusion, controlled manuallyby a six-way rotary valve. Intracellular Voltage Recordings-Intracellular recordings of membrane potential were made at 35-37 'C using a WPI model FD-223 electrometer and glass electrodes, which when filled with 1 M KC1 had resistances of100-150 M. Cellswere continuously superfused with bath solution containing (in mM): 140 NaCl, 5 KC1, 2 MgC12, 2 CaCl2, 10HEPES, and 5 glucose, pH 7.4.
40
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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 detailelsewhere,' and a novel non-inactivating current, which in view of its location incells of the adrenal cortex, we have named IAC. During prolonged recordings, IACamplitude increased dramatically from3.73 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,IACwas clearly distinguished from A-type current by its voltage-independent availability. At a holding potentialof -40 mV, the transient K+ current was completelyinactivated while IACwas unaffected. When IACwas viewed in isolation under these conditions,no delay incurrentonsetwasapparentwith depolarizing steps to potentials up+10 to mV, suggesting that IAC channels remain open at the holding potential (Fig. 1B). Current-voltage relationships obtained from several cellsthat 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. K+ channel antagonists, IAC Although blocked by several 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 byvoltage steps to +20 mV almost completely, but reduced IAC 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 wasinsensitive to wdendrotoxin and apamin at concentrations up to500 nM, but disappearedcompletely 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.
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POTENTIAL (mV)
FIG. 1. Characteristics of IACin bovine AZF cells. A , timedependent growth of IAC. K+ currents were activated at 30-5 intervals byvoltage steps to +20 mV from a holding potential of -80 mV. Current records at indicated timesafter initiating whole-cell recordings. B and C, current-voltage relationshipsfor 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, IACwas 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 resistanceerror, 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 intracellularfactor affected by cell dialysis. Thetime-dependent increase inIAc 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 IACby a GTP-binding protein. We further tested thispossibility by preincubating cells with bacterial toxinsfrom Vibrio cholera (CTx) andBordatella pertussis (PTx), which, respectively, activate Gs and suppress activation of Gi and Go. Functional expression of IACwas affected only by CTx, which completely inhibited its appearance, indicating that IACwas 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 IAC might be modulatedby peptides that physiologically regulate cortisol production, AZF cells
ACTH and AII Inhibit a Novel K" Current in
8642
Adrenal Cells
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FIG. 2. Modulation of IAC expression by guanine nucleotides and toxins. The time-dependent amplitude ofIAc was measured as described in Fig. 1A with pipette solutions containing 200 PM GTP as control (n = 45),no GTP ( n = 5), 0.5-1 mM GDPPS (n = ll),or 0.2 mM GTPrS (n = 11).Alternatively, cells were treated for 6-12 h -14 with 200 ng/ml PTx ( n= 9) or for 5-10 h with 1 pg/ml CTx ( n= 11) -13 -12 -11 -10 -9 -8 -7 -6 CONCENTRATION (log M) before recording currents as in A . IAC was measured as the noninactivating current present at the endof the test pulse. Results are FIG. 3. Time and concentration-dependent inhibitionof IAC mean 2 S.E. of initial ( A ) and maximal ( B ) IACcurrent densities by ACTH and AIL After voltage clamping cells at -80 mV, test determined for each cell bydividing IACamplitude by cell capacitance. pulses to +20 mV were applied at 30-5 intervals from a holding IACreached maximum amplitudes after 20-60 min of recording. potential of -80 mM. A, selective block of L C by ACTH. Current records before and after superfusion of cell with 10 p~ ACTH. Top were exposed to ACTH and AI1 while recording K+ currents. two traces, current records immediately before and 2 min after conIAc was potently and selectively inhibited by both peptides. tinuous superfusion of cell with ACTH. Middle and bottom traces, 3 and 4 min after superfusion of ACTH. Note: bottom truce is superACTH produced complete inhibition of IACwith an ECm of imposed on control trace obtained immediately after establishing 4.1 PM (Fig. 3, A and C). The current was inhibited equally whole-cell clamp. B, time-dependent inhibition. Fraction of unat potentials from -80 to +40 mV (Fig. IC). AI1 was also blocked current is plotted against timefor cells exposed to either 100 effective at subnanomolar concentrations (EC, = 145 pM), PM ACTH ( n= 11)or 10 nM AI1 ( n = 6). Values are mean f S.E. c, but IAc was reduced by a maximum of 77.5 f 2.8% (Fig. 3C). concentration dependence. Fraction of unblocked IAC is plotted against peptide concentration for ACTH and AI1 as indicated. Values The inhibition ofIAc by both peptides began after a delay of are mean f S.E. of 4-12 separate experiments.
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 IAC 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 patchcontaining 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 patchwith 1 nM ACTH, channel opening had ceased (Fig. 4, B and D).The currentvoltage relationship indicated that theACTH-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.
Intracellular voltage recordings showed that bovine AZF cells maintained resting potentials of -71.1 f 1.1 mV ( n = 65) and were depolarized by changing external K+ concentrations as predicted by the Nernst equation for a membrane selectively permeable to thision only. ACTH depolarized cells by a maximum of 49 & 1.8 mV with an ECWof 10.4 p~ (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 andwas 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,
8643
ACTH and AII Inhibita Novel K+ Current inAdrenal Cells A
FIG.4. Effect of ACTH on single channel currents. Outside-out patch
recordings were made at +20 mV before and after superfusingthe patch with aaline containing 1 nM ACTH. A and B, all points amplitudehistogramsconstructed from 48 consecutive 400-ma sweeps (97,152 total points), before ( A ) and 5 min after ( B ) exposing the patch to 1 nM ACTH.Cutofffrequencyis 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 containingone channel at various test potentials from -40 to +50 mV. Points are mean & S.E. of 4-26 measurements. A unitary conductance of 63 picosiemens was determined by least square linear regression between +20 and +50 mV. Pipette and in reexternal solutions aswhole-cell cordings.
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been reported to modulate both Ca2+ and transient K+ currents in rat and bovine adrenal glomerulosa cells (3, 5, 17, 18), these peptides did notaffect T3 or A currents in bovine AZF cells. ACTH and AI1 are known to act on adrenal corticalcells through separate signaling pathways. ACTH stimulates the synthesis of CAMP, whereasAI1 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 thatproduce no measurableincrease in cAMP (19, 20). Thus it appears unlikely that either of these effects r _CONTROI is mediatedby this second messenger. In thisregard, previous studies on adrenalcells have led to theconclusion that cAMP is the primary intracellular mediator of ACTH-stimulated steroidogenesis (21-23). This notion remains even though ACTHtriggers Ca2+ uptakeandstimulates corticosteroid production at concentrations far lower than those required to T i P i (msn) (8, 10, 19, 24). The excellent produce increasesincAMP FIG.5. Depolarization of AZF cells by ACTH and AII. Con- correlation between ACTH-stimulated inhibition of IACand centration dependence and temporal pattern are shown. After impal- membrane depolarization with Ca2+ influx and corticosteroid ing a cell and obtaining a stable resting potential, cells were super- secretion suggest that Ca2+ may be the primary intracellular fused with ACTH(1-24) or AI1 at various concentrations while continuously recording membrane potential. A , concentration messenger, particularly a t low ACTH concentrations. dependence; maximum depolarizationis plotted against peptide con- Activation of phospholipase C by AI1 enhances the synthecentration. Values are mean & S.E. of 5-9 separate determinations. sis of inositol 1,4,5-trisphosphate anddiacylglycerol and trigB and C , time course and reversal of depolarization by 50 p M ACTH gers the release of Ca2+ from intracellular stores. Whether peptides and control and 250 p~ AII.Cellsweresuperfusedwith one of these second messengers inhibits IAC is not known. solution at the indicated times. Although Ca2+-mediated inhibitionof K+ channels has been bovine AZF cells express a low voltage-activated T-typeCa2+ observed in several cell types (14, 25, 26), it is unlikely that current (15). We propose that these channels determine the Ca2+ released from intracellular stores inhibited IAC in our electrical properties of AZF cells and transduce biochemical experiments since intracellular Ca2+ was highly buffered by signals originating at the cell membrane to depolarizationBAPTA. dependent Ca2+ entry andsecretion. Specifically, ACTH and Our results demonstrating inhibition of IAC by GTP+ and AI1 acting through separate receptors inhibit IAC, triggering CTx indicate that the corresponding channels are under the the sequence of membrane depolarization, Ca2+channel acti- inhibitory control of a Gs protein. This may be the first vation, and corticosteroid production. In thisregard, we have outwardly rectifying K+ channel to be inhibited by this GTPfound that T-type Ca2+ channel antagonists block ACTH- binding protein.An outwardly rectifying K+ current, which is and AII-stimulated cortisol production and Ca2+ current a t present at negative potentisls and modulated by G-protein similar concentrations (15).3Although ACTH and AI1 have activation, has been identified in a leukemic cell line (16). In J. J. Enyeart, B. Mlinar, and J. A. Enyeart, submitted for publi- spite of some resemblance to IAC,the current inleukemic cells cation. differs in several important respects. In particular, the func-
8644
ACTH and AII Inhibit a Novel K+ Current inAdrenal Cells
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 throughaG-proteinintermediate 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 IACmaximally in cells that have been patch-clamped in the whole-cell modefor periods of 1 h. Responses requiring the synthesis of diffusible second messengers wouldlikely have been “washed out” long before this time. Regardless of the signal transduction pathways involved, the convergent inhibition ofIAc 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. REFERENCES 1. Capponi, A. M., Lew, P. D.,Jornot, L., and Valloton, M. B. (1984) J. Biol. Chern. 269,8863-8869
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