Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosa cells STEPHEN J. QUINN, ULRIKE BRAUNEIS, M. CARTER CORNWALL, AND GORDON
DOUGLAS L. TILLOTSON, H. WILLIAMS
Endocrine Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston 02115; and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118 Quinn, Stephen lotson, M. Carter
J., Ulrike Cornwall,
Brauneis, Douglas L. Tiland Gordon H. Williams.
Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosacells.Am. J. Physiol. 262 (Cell Physiol. 31): C598-C606,1992.-Rat and bovine adrenal zona glomerulosa (ZG) cells possessa low-threshold, voltage-dependent Ca2’ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membranepotentials above -80 mV with maximal peak Ca2’ current elicited near -30 mV. Inactivation of the Ca2’ current was half-maximal between -74 and -58 mV, dependingon the external Ca2’ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Ca;) levels.Under basalconditions, Ca; is stable in single rat ZG cells, whereasmore than half of the bovine ZG cells produce repeated Cai transients. These Ca; transients, which are blockedby removal of external Ca2’ or addition of Ni2+,are likely dueto repetitive electrical activity in bovine ZG cells. Ca; responsescan be elicited by small increasesin external K’ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+channels. However, these Cai changes remain robust at high external K’ concentrations (20-40 mM). In experiments combining Ca; measurementsand wholecell voltage clamp,a steepdependence of Cai on membranepotential wasrevealed beginning at depolarizing voltages near a holding membranepotential of -80 mV. A maximal increasein Cai occurred near -30 mV (equivalent to an external K’ concentration of 40 mM), a membrane voltage at which sustainedcurrent through low-threshold Ca2+ channelsshould be negligible. These data raise the possibility of additional voltage-dependentpathways for Ca2+influx. calcium current; adrenal
brane sufficiently
to activate this conductance
(11, 17,
20, 22-24).
Critical to the hypothesis that this Ca2+ conductance is involved in the control of Cai and steroidogenesis is the relationship between membrane potential and the voltage dependency of channel activation and inactivation. Small depolarizations from the resting membrane potential, as observed with millimolar increases in external K’, must significantly activate the Ca2+ conductance. In addition, the channel current must support a sustained increase in Ca2+ influx and accumulation during secretagogue stimulation. Previous studies have shown that elevation of external K+ stimulates an increase in Cai with a dose dependency similar to that for aldosterone production (4, 26). However, there remain questions as to the duration of the Cai change by elevation of external K+ with implications for the kinetics of Ca2+ channel activation and inactivation (1, 10). Moreover, previous studies examining the electrical properties of bovine and rat ZG cells indicate differences between these species that make a comparison of their Ca2+ current(s) and external K+-induced Cai changes important for our understanding of the human adrenal cortex (5, 6, 18, 19, 22, 23). MATERIALS
AND METHODS
IS AN IMPORTANT REGULATOR ofsteroidogenesis in zona glomerulosa (ZG) cells (1, 25). The three major secretagogues, external K+, angiotensin II (ANG II), and adrenocorticotropic hormone (ACTH), require external Ca2’ for aldosterone stimulation and may increase Ca2+ influx to produce a rise in cytosolic-free calcium (Cai) (15, 16, 25-27, 29). While it has been difficult to observe elevation of Cai in ZG cells during ACTH stimulation (16, 29), sufficient data exist to postulate that increases in Ca2+ influx and Cai serve as central elements of the transduction mechanisms of secretagogue stimulation. Recent electrophysiological evidence points to the activation of voltage-dependent Ca2+ channels as a common pathway for Ca2+ entry into ZG cells during stimulation. Membrane voltage and current measurements demonstrate the existence of a Ca2’ conductance in rat and bovine ZG cells (5, 6, 18, 19, 22, 23), and each major secretagogue can depolarize the mem-
Adrenal cellpreparation. Experiments were performed on ZG cellsisolatedfrom rat and bovine adrenal glands.ZG cellsfrom Squague-Dawleyrats were prepared by techniques previously reported (30). Calf adrenalswere obtained from a local slaughterhouse. The adrenalswere sliced with a microtome and decapsulatedusing scissorsand scalpel.The remaining capsular tissuewas placed in a Krebs-Ringer bicarbonate (KRB) solution supplemented with 2% glucose and 4% bovine serum albumin (BSA) and containing crude collagenase(3.7 mg/ml) and deoxyribonuclease(0.05 mg/ml), mincedwith scissorsand incubated for 35 min. The tissue was resuspendedand incubated for an additional 30 min with collagenase(3.7 mg/ml). The supernatant and remaining tissuewere filtered through a 210-micron nylon mesh. After three washesin KRB, the cell suspensionwas placed in a superfusionchamber mounted on the stageof an inverted microscope.The bottom of the chamber consistedof a glasscover slip, which had been coated with a 0.1% solution of commercial gelatin (Knox, Englewood Cliffs, NJ) and treated with a 1 M NaCl solution containing 0.2% concanavalin A (Sigma). The cellsadheredto the glasssurface without apparent adverseeffects to cell viability or membrane properties. The bath chamber was continuously perfusedwith warmed(35-38°C) solution (120mM NaCl, 4 mM KCl, 1.2mM Na2HP04, 1.25 mM CaC12,0.5 mM MgS04, 20 mM HEPES, 0.1% BSA, and 0.2% glucose;pH = 7.4). For experiments combining microspectrofluorimetry and whole cell voltage clamp, cells were plated on glasscover slips
C598
the American
CALCIUM
0363-6143/92
$2.00
Copyright
0 1992
Physiological
Society
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cA2+
CHANNELS
AND
CYTOSOLIC
and placed in 60-mm culture dishes. The cells were incubated for 1 to 3 days prior to use. This brief period of culturing makes the formation of gigaohm seals more feasible. The ability of external K+ to stimulate steroid secretion is retained during this time. ELectricaL recordings. The whole cell recording technique was used to voltage clamp the ZG cells (9). Glass pipettes were pulled from Corning 7052 and 8161 capillary tubing with a twostage puller and firepolished to a pipette resistance of 2-10 MQ when filled with internal solution. The pipette was connected to the head stage of a patch-clamp amplifier (List, Darmstadt, FRG), which was mounted on a Huxley manipulator (Custom Medical Research Equipment, Glendora, NJ). Capacitance and series resistance compensation was adjusted to optimize the speed of the voltage clamp. The electrical signals were low-pass filtered at 1.3-3 kHz and displayed on a storage oscilloscope. Signals were digitized and stored with an IBM PC computer for later analysis. Currents were leakage corrected by subtraction of the scaled linear leakage current measured during small hyperpolarizing and depolarizing voltage steps. When using high concentrations of divalent cations, adding of potassium channel blockers, or substituting for Na+, equimolar concentrations of NaCl were replaced. Standard composition of the internal pipette solution for measurement of Ca2+ current was the following (in mM): 140 CsCl, 5 NaCl, 2 MgC12, 1 EGTA, 5 Na2ATP, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES); pH = 7.2. For experiments that investigated total whole cell current, CsCl was replaced by KCl. Measurement of Cai. The measurement of Cai is similar to methods previously described (26, 27). Dispersed ZG cells were incubated for 20 min with fura-2/acetoxymethyl ester (AM) (Molecular Probes) at a concentration of 5 PM in an Eagle’s minimum essential medium (MEM) including 20 mM HEPES, 1.25 mM CaC12, 0.5 mM MgSO,, and 0.1% BSA; pH = 7.47. Cell suspensions were washed and used l-4 h following loading with dye. Test solutions consisted of the standard bath solution and varying concentrations of external K’ with an equimolar adjustment of NaCl. As described previously, the test solutions were applied by puffer superfusion, which allows rapid, local solution changes around an identified ZG cell (24, 26, 27). The whole cell Ca; measurement system consisted of a dual wavelength light source (Photon Technology International), an inverted microscope equipped for ultraviolet fluorimetry (Nikon Diaphot), and a photomultiplier tube (PTI). Furawas used to estimate Ca; by the ratiometric method. Excitation wavelengths were centered at 350 and 380 nm with a bandwidth of 4 nm. Calibration constants were determined by an in vitro method and a dissociation constant (&) of 224 nM was used, as previously reported (8). Estimated Ca; concentrations fell within a range where fura- reliably monitors Cai. Measurement of Ca; during whole cell voltage clamp. Ca; was measured in single ZG cells while under whole cell voltage clamp. A digital video imaging system was used to monitor Ca; as described previously (26). Furawas introduced into the cells via the patch pipette by adding 500 PM of the fluorescent probe to the patch pipette solution containing the following (in mM): 124 KCl, 4 NaCl, 1 Mg-ATP, and 20 HEPES. Fluorescence images were digitized, and the emission signal from an entire cell was integrated and stored as a single value for each excitation wavelength at each time point. This operation was performed in real time at a sampling rate of 2 Hz. Identification of zona glomerulosa cells. Cells were identified as ZG cells based on morphological characteristics described by Tait et al. (28). Cells were spherical with a diameter of 7 to 12 pm. The nucleus was round and occupied much of the cell volume, and the cytoplasm contained numerous small lipid droplets. Data analysis. Ca; responses were analyzed by one-way analysis of variance and the Newman-Keuls multiple comparison
cA2+
IN
ADRENAL
ZG
c599
CELLS
test. All Ca; recordings were digitally Savitsky-Golay smoothing routine.
filtered
with
a U-point
RESULTS
Whole cell current. Rat ZG cells have a dominant K+ conductance at resting and depolarizing membrane voltages (23). Consistent with this finding, a significant outward current is seen during whole cell current recordings under conditions that simulate the physiological ionic composition of the external and internal solutions. Figure IA shows the current response to step changes in voltage from a holding potential of -100 mV. At voltages more positive than -80 mV, both early inward and more slowly developing outward currents were elicited. In Fig. lB, the current-voltage relationship illustrates the voltage dependency of the early and late current. As the membrane voltage was stepped to more positive values, substantial outward current was observed and early net inward current was no longer seen. Potassium channel blockers [Cs’, tetraethylammonium (TEA) and 4-aminopyridine (4-AP)] inhibited the outward current. These results are similar to those we have previously described for outward current in rat ZG cells (3). Inward calcium current. To isolate the inward current from the outward current, K+ was replaced by Cs+ in the internal solution, and, for some experiments, TEA and 4-AP were also added to the bath solution. As seen in Fig. 2A, only inward current was evident following depolarizing voltage steps when the cell was dialyzed with Cs+ internal solution. In bath solution containing 2.5 mM Ca’+, the threshold for activation, the membrane voltage at which active current is first measurable, was between -80 and -70 mV, with maximal peak inward current occurring near -30 mV (Fig. 2B). In other experiments, a decrease in the peak current was found at membrane voltages more positive than 0 mV, but reversal of the inward current was not reached. Substitution of external Na+ by tris( hydroxymethyl)aminomethane (Tris) did not appreciably alter the inward current, implying that Na’ was not significant current carrier. Typically, the inward current in ZG cells was larger in external solutions containing higher Ca2+ concentrations, suggesting that the current was principally carried by Ca2+ (Figs. 2 and 5). Current-voltage relations tended to shift in the depolarizing direction when higher Ca2+ concentrations were used, with the threshold voltage lying between -60 and -50 mV for 10 mM Ca2’. Mn2+ (5 mM; n = 3) or Cd2+ (1 mM; n = 3) blocked the inward current, as has been seen for other Ca2+ currents (data not shown). The inward current displayed strong voltage dependence for activation and inactivation (Fig. 2). The timeto-peak was sharply reduced as the depolarizing voltage step was increased to more positive values. Likewise, substantial inactivation occurred during voltage steps of lOO- to 200-ms duration. Figure 3 shows that the inward current demonstrated similar kinetics and current-voltage relationship when carried by Ba2+. Current inactiuation. Current inactivation was studied by holding membrane voltage at different levels prior to presenting a test voltage step. Figure 4A shows currents elicited in 2.5 and 10 mM Ca2+. The peak current de-
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C600
cA2+
A
CHANNELS
AND CYTOSOLIC
cA2+
B
-SO-'\---
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-70*-
-80 A
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voltage, mV -60/ -?-T
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ZG CELLS
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IN ADRENAL
Fig. 1. Whole cell current from a single rat ZG cell. A: current responses to voltage steps that are marked to left of each trace. External solution contained 2.5 mM Ca2+. B: current-voltage relationship plotted as peak early current (filled circles) and current at end of voltage step (open circles). Current recordings were leakage corrected. Holding potential, -100 mV. Internal solution contained KC1 as principal ion species.
- -200
creased as the holding potential was varied from -100 mV to more depolarizing values. Peak current was normalized to the peak inward current observed when the membrane voltage was held at -100 mV and was plotted vs. the holding potential in Fig. 4B. Data were fitted to the equation (1 ) h = [l + exp (V - K-J/k]-’
cells. The inward currents of both rat and bovine ZG cells could be isolated from outward currents with Cs+ in the internal solution. Figure 5 compares the inward currents in the presence of 10 mM external Ca2+. The kinetics of the inward current were nearly identical for time-to-peak and the time course of inactivation (Fig. 5A). In Fig. 5B, current-voltage relationships are plotted for both rat and bovine ZG cells. The analysis of current was identical to that described above. To where h represents the inactivation normalized to the inactivation maximal peak current, Vos5is the membrane voltage better compare the current-voltage curves, the peak current at each membrane voltage was normalized to the where h is half-maximal, V is the holding membrane maximal peak current, which was elicited by a voltage voltage, and k is a slope constant. The voltage dependency of this inactivation was steep (k = 4.0) with a Vo.5 step to -20 mV in both examples. Most apparent was of -73 mV for 2.5 mM Ca2’ and -58 mV for 10 mM the similarity of the current-voltage relationship (Fig. 5B), indicating the same voltage dependency for the Ca2+. Comparison of calcium current in bovine and rat ZG inward Ca2+ current in both species.
B
A 2.5 mM Ca2+ -70
membrane -80
-60
-40
potential,
-20
mV
20
0
-iv
-60 -40
'200
-20* 0 Y
1
v
1
100 msec
1
2 g 2 4 gct
Fig. 2. Inward current from rat ZG cell. A: traces represent inward current elicited by voltage steps in 2.5 mM Ca2’. Voltage steps are noted to left of each record. B: current-voltage curve for peak inward current in 2.5 mM Ca2’. Current traces were leakage corrected. Holding potential, -100 mV.
;b *
t-
J 125 pA
J
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CHANNELS
cA2+
AND
CYTOSOLIC
cA2+
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ADRENAL
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CELLS
C601
A membrane
5 mM Ba2’
-60
-40
potential,
mV
-20
0
20
f
B
200 5 z
O/$
50 msec
Fig. 3. Inward current carried by barium. A: traces represent inward current elicited by voltage steps in 5 mM Ba2’. Voltage steps are noted to left of each record. B: current-voltage curve for peak inward current in 5 mM Ba2’. Current traces were leakage corrected. Holding potential, -80 mV.
4
mM and diminished substantially with 40 mM to a level roughly equivalent to the Cai response at 10 mM external K+. The mean amplitude change at each external K+ concentration was larger for the rat than the bovine, consistent with the larger Ca2+ current found in rat ZG cells. The increase in Cai stimulated by 10, 20, and 40 mM external K+ was completely blocked by 1 mM Ni2+ or 1 mM Cd2+ (data not shown). Lower concentrations of these divalent cations produced partial inhibition. These results are similar to those we have previously described for Cai responses to smaller elevations of external K+ (26) Membrane voltage and Cai changes. Increases in exter‘cai responsesto external K+ in rat and bovine ZG cells. As observed in previous studies, Cai levels in ZG cells nal K+ concentration can depolarize the membrane powere exquisitely sensitive to changes in external K+ tential to a new value, analogous to the voltage-clamp concentration. Elevation of external K+ from a basal technique. In rat ZG cells, elevation of external K+ value of 4 mM to concentrations ranging from 6 to 40 concentration by the puffer pipette method can produce that is complete within 100 mM produced prompt increases in Cai (Fig. 7). At low a membrane depolarization concentrations of external K+, the Cai change was sus- ms and sustained for the duration of stimulation (data tained with little change in amplitude in rat ZG cells; not shown). Voltage clamp offers even faster and more precise control of membrane voltage and eliminates any however, Cai transients were often found under similar conditions in bovine ZG cells. Of the bovine ZG cells additional effects resulting from the elevation of external tested with stimulation by 6 mM external K+, over 50% K + To directly evaluate the relationship between memdisplayed rapid, repeated Cai transients that had a greater frequency and/or amplitude than under basal brane voltage and Cai, techniques were combined to monitor Cai during a series of voltage steps while the conditions of 4 mM K+. At high external K+ concentrations, the Cai change was typically stable in rat and individual ZG cell was placed under whole cell voltage bovine cells, although a slow decline from maximal Cai clamp (Fig. 9). As the magnitude of the depolarizing levels was observed in some cases. More than 90% of the voltage step was increased, the change in Cai also inbovine and rat ZG cells displayed a Cai response when creased with a threshold voltage between -80 and -70 subjected to high external K+ concentrations. mV to a maximal response at -31.43 t 4.25 mV (n = 8). The dose-response relationships (Fig. 8) for Cai re- Further depolarization progressively reduced Cai values, sponses stimulated by external K+ indicate that the resulting in a 44 t 15% (n = 8) decrease in the emission amplitude of the Cai change rises steeply between 4 and ratio change with voltage steps to values between 0 and 10 mM external K+ in both rat and bovine ZG cells. At +4O mV. Longer voltage steps to voltages that produced higher external K+ concentrations, the Cai peaked at 20 near-maximal Cai changes demonstrated a sustained Cai Basal Cai levels in rat and bovine glomerulosa
cells. Cai
was monitored in single ZG cells isolated from rat and bovine adrenals. The mean basal Cai was similar for rat, 98 t 4 (SE) nM (n = 258), and bovine, 113 t 9 nM (n = 45), ZG cells. Basal Cai was defined as the level between oscillations. Basal Cai was stable in rat ZG cells, but over 50% of bovine cells displayed spontaneous Cai transients (Fig. 6). When these Cai oscillations were present in bovine ZG cells, they tended to have a consistent amplitude for each individual cell. These basal Cai transients were completely inhibited by ion substitution of Mg+ for external Ca2+ or by the addition of 1 mM Ni2+ (Fig. 6)
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C602
CA2+ CHANNELS
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+
-
-60-J-0----
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-7oiL_-
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ZG
CELLS
Cd2+, and Ni2+ substantially block it. Threshold voltage for activation lies between -80 and -70 mV in superfusates containing Ca2+ concentrations (254.0 mM) close to the physiological range. Significant inactivation is observed during sustained depolarizations, which is largely dependent on voltage rather than Ca2+ entry, because the kinetics of inactivation are observed in the presence of a strong Ca2+ chelator [ 1 mM ethylene glycolbis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA)] in the patch pipette and are retained when
mM Ca2'
10
cA2+ IN ADRENAL
CYTOSOLIC
-
A rat I pre-pot. iI1
msec ‘J
100
5
bovine
10 mM Ca”
10 mM Ca”
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pre-potential,
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rat ZG cells. A: currents elicited by Ca2’ and -30 mV for 10 mM Ca2’ (pre-pot) shown to left of each trace. for 2.5 mM Ca2’ and 5 s for 10 mM Ca2+. Data were from 2 different rat ZG cells. B: voltage dependency of inactivation. Peak inward currents for a voltage step to -40 mV were normalized to peak current value elicited when holding potential was -100 mV (ha) and plotted vs. pre-potential value. Smooth curves through data points were derived from &. I with the Vo.5 of -73 mV for 2.5 mM Ca2+ (circles) and -58 mV for 10 mM Ca2’ (squares) and k value for 4.0.
plateau achieved within the first 5 s and lasting for the pulse duration of 20 s (data not shown). Simultaneous measurements of inward Ca2’ current and Cai were not possible as the outward current was not inhibited. An external Ca2+ concentration of 1.25 mM was used to approximate physiological conditions. DISCUSSION
voltage-dependent
0 0 4
100 msec
Fig. 4. Current inactivation from a voltage step to -40 for 2.5 mM from different holding potentials Pre-potentials were held for 10 s
A similar
4 a
0” Lo
Ca2+ current is found in
ZG cells of rat and bovine adrenal tissue that appears
responsible for increases in Cai stimulated by small physiological elevations in external K+ concentration. The membrane conductance change giving rise to this current is selective for divalent cations. Both Ca2’ and Ba2’ are carried through this conductance pathway, while Mn2+,
s
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Fig. 5. Comparison of rat and bovine zona glomerulosa cells. A: traces represent inward current elicited in 10 mM Ca2+ by voltage steps, indicated to left of each record. Note that current calibrations are different for each ZG cell. Current records were leakage corrected and holding potential was -80 mV. B: current activation: peak inward current was normalized to maximal value elicited with a voltage step to -20 mV and plotted on right-hand side of graph (rat: open squares; bovine: open circles). Maximal peak current was 750 and 125 pA for rat and bovine ZG cells, respectively. Current traces are shown in previous figure. Current inactivation: peak inward currents for a voltage step to -30 mV from different holding pre-potentials were normalized to maximal value elicited with a holding potential of -100 mV and plotted on left-hand side of graph (rat: filled squares; bovine: filled circles) vs. pre-potential value. Smooth curves through data points were derived from Eq. 1 with Vo.5 = -58 mV and k = 4.
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CHANNELS
CA2+
CYTOSOLIC
“0” Cd +
r
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cA2+
bovine
bovine
2
AND
i t
1 mM
Ni2+
ADRENAL
ZG
100 400
,“bb’nwf .ff
.
Fig. 6. Basal cytosolic calcium (Ca;) and effects of nominal zero calcium and nickel in bovine zona glomerulosa cells. Shown are representative basal recordings from 4 individual bovine ZG cells. Roughly half of bovine ZG cells exhibited basal Cai transients. Timing of external calcium removal or addition of 1 mM nickel is indicated by arrows. External Ca2’ concentration, 1.25 mM.
M&p%%&
44
100
100
I
I 1 minute
inactivation are more negative than in many other reports of low-threshold Ca2+ currents, which is possibly due to the high divalent cation concentrations typically employed when evaluating Ca2+ currents. Another interesting feature of the inward current of ZG cells is the predominance of low-threshold Ca2’ current over other types of Ca2+ current. In most other cell types, the highthreshold, L-type Ca2+ current is much larger than the low-threshold Ca2’ current. The magnitude of the lowthreshold Ca2’ current may, in part; be responsible for the unusual sensitivity of ZG cells to external K+ concentration, where very small membrane depolarizations
external Ca2+ is replaced by Ba2+. Our experimental conditions did not allow for the direct evaluation of Ca2+dependent inactivation. The Ca2’ current available for activation is dependent on the membrane potential. Half-maximal current inactivation was observed between -73 and -58 mV, with the most negative values found at external Ca2’ concentrations closeto the physiological range, as reported in a previous study using rat ZG cells (18). Therefore, the Ca2’ current found in ZG cells resembles the low-threshold, T-type Ca2+ current characterized in many other tissues (2, 7, 31). In our experiments, the voltage dependences of activation and of rat
C603
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CELLS
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1
1500-
40 mM
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I
500-
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40 mM .
OL
----..A 1
1
1
1
1
0
I
10 20 mM Fig. 7. Time course of Cai response to different external potassium concentrations in rat and bovine zona glomerulosa cells. Shown are representative Cai recordings from 4 different rat and bovine ZG cells during puffer superfusion with elevated K+. Bar above the Cai recordings indicates onset and termination of external K+ elevation. Concentration of external K+ is indicated for each pair of traces. External Ca2’ concentration, 1.25 mM.
10 mM
0‘
1 0
.
1
1
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1 0
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C604
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CHANNELS
AND
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CELLS
f
I
II
1
I
I
I
4
56
8
10
20
40
external
potassium,
mM
Fig. 8. Relationship between maximal Cai change and external potassium concentration. Maximal Cai change is increase above basal levels when external K+ concentration is elevated from a basal value of 4 mM. Each data point represents mean t SE from at least 10 individual rat or bovine ZG cells at each external K’ concentration.
can lead to substantial increases in Ca2’ influx, Cai, and hormone secretion. The Cai current described in this study is consistent with our previous voltage recordings of Ca2+ action potentials characterized in rat ZG cells (23). The thresholds for activation and current inactivation have similar voltage dependency, and the selectivity and blockage of the underlying conductance are the same. Other recent studies using voltage-clamp techniques have also demonstrated Ca2’ currents in rat and bovine ZG cells. Matsunaga et al. (18) found only a transient Ca2+ current with a low-threshold for activation in freshly dispersed rat ZG cells, while other investigators have reported as many as three different Ca2’ currents in cultured rat ZG cells (6, 22) with voltage dependencies similar to the T-, L-, and N-type calcium channels of the chick sensory neuron (21). The reason for differences among these studies is unknown but may involve the use of freshly dispersed vs. cultured ZG cells. Studies using bovine ZG cells have consistently found two types of Ca2+ current (5,19). The low-threshold current had properties similar to that described in our study and was found in all bovine ZG cells, while a high-threshold Ca2+ current similar to the L-type was reported to be present in a small percentage of the cells (5). Adrenal ZG cells also possessvoltage-dependent outward currents that may be important in the stimulation of aldosterone production by its major secretagogues (3). The outward current is likely to be carried by K+ because it follows the ionic gradient for K+ across the plasma membrane and is blocked by replacing K+ by Cs+ in the internal solution. Other studies have also demonstrated K+ conductances in ZG cells both at resting membrane potential and with membrane depolarization (3, 22, 23). The major secretagogues induce membrane depolarizations either by reducing the driving force for K+ (elevation of external K’) or by blocking one or more of these K+ conductances (ANG II and ACTH) (3, 20, 22-24). This membrane depolarization may be a common transduction mechanism for the stimulation of Ca2+ influx
1
I
0
100
A
L
200 time, set
300
B 0.4
0.3 & s A0 0
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.dg .dsi E Q, 0.1
0 1
-80
A
I
-40 membrane
A
1
0 voltage,
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Fig. 9. Relationship between membrane voltage and Cai concentration. A: top trace shows change in ratio of light emission stimulated by excitation wavelengths of 350 and 380 nM, respectively. Bottom trace indicates timing and amplitude of a 2-s voltage step applied from a holding potential of -80 mV to a bovine ZG cell under whole cell voltage clamp. Interval between voltage steps varied to allow return to basal Cai values. B: plot of maximal change in emission ratio vs. membrane voltage. Change in emission was corrected for rundown of Cai response during course of experiment by comparing Cai responses to equivalent voltage steps to -20 mV applied at different times during experiment and assuming a linear decay of Cai response over time. Similar results were found for bovine (n = 7) and rat (n = 1) ZG cells. External Ca2’ concentration, 1.25 mM.
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CA2+
CHANNELS
AND
CYTOSOLIC
cA2+
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ADRENAL
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CELLS
C605
produce significant changes in Cai. Previous studies of through the opening of voltage-dependent Ca2+ channels. Thus the low-threshold Ca2+ current is likely to play an membrane voltage and Cai responses in rat ZG cells are important role in the regulation of Cai levels and aldosteconsistent with the hypothesis of a sustained Ca2+ current through low-threshold channels. rone production for a number of its secretagogues. From our previous work, rat ZG cells were found to Large changes in external K+ concentration also prohave an average resting membrane potential of -78 mV duce sustained Cai responses in bovine ZG cells. Under at an external K+ concentration of 4 mM. The relationthese conditions, bovine ZG cells may also utilize a ship between membrane voltage and external K+ was 45 sustained calcium conductance to produce stable Cai signals. This is substantiated with Cai measurements mV voltage per lo-fold change in external K+ concentration (23). Because the threshold voltage for Ca2+ channel under voltage-clamp conditions when voltage steps of 5activation and the membrane voltage for half-maximal s duration produce a continuously increasing Cai response likely due to a sustained Ca2+ current. Moreover, inactivation lie close to the resting membrane potential, sustained current through these Ca2+ channels should voltage pulses of 20-s duration elicited sustained Cai only occur with small membrane depolarizations such as changes that rapidly returned to basal levels with the the 7-mV depolarization predicted for an external K+ termination of the voltage step. Had Ca2+ current completely inactivated, a decline of Cai would have been elevation from 4 to 6 mM. This hypothesis is consistent expected during a sustained pulse. These Cai data can be with the steep dependence of Cai level and aldosterone production on external K+ concentration between 4 and contrasted with Ca2+ current recordings, which show 10 mM (5-7). Likewise, experiments using voltage pulses near complete inactivation by the end of a 200-ms voltage significant in- step, indicating the sensitivity of Cai as an indicator of to activate Ca2+ current demonstrate creases in Cai with voltage steps from -80 to -70 mV. maintained Ca2+ influx. Stimulation by external K+ or direct membrane voltage Some features of Cai responses during stimulation with high K+ concentrations or large positive voltage steps change elicits equivalent Cai responses, pointing to voltage as the primary transduction element for external K+. cannot be easily explained by a sustained conductance through low-threshold Ca2+ channels. At membrane voltOther effects of increasing the concentration of external K+ probably do not contribute to the Cai or aldosterone ages more positive than -50 mV (external K+ concentrations greater than 20 mM), sustained Ca2+ current responses. Questions remain as to the mechanism for sustained through these channels should be vanishingly small, yet secretagogue-induced Ca2+ influx and the sustained rise the Cai responses reach their peak amplitudes in this voltage and external K+ range. Two possible explanaof Cai observed with K+ stimulation in rat and bovine cells. The present findings suggest two possible ways by tions for these disparate results are the following. First, which Ca2+ influx may remain elevated during long-term analysis of low-threshold Ca2’ current may not accusecretagogue stimulation. First, the transient Ca2+ cur- rately describe the sustained current through this conrent may be repeatedly activated by membrane depolarductance system. There may be inaccurate determinaization resulting from electrical activity. Trains of spike tions of the voltage dependency of the sustained Ca2’ activity have been observed in some cells from kitten current because the current amplitudes are very small in adrenal capsular tissue at rest and during K+- or ANG the voltage range of interest and external Ca2+ concenrange were used in II-stimulated depolarization (20). Information on elec- trations greater than physiological trical activity of bovine ZG cells is not available. How- the electrophysiological measurement of the Ca2+ curever, bovine ZG cells exhibit repeated Cai transients rents. Alternatively, the voltage dependency of current inactivation may be significantly different when memduring basal and membrane depolarized conditions. Thus bovine ZG cells may repeatedly activate the transient, brane voltage is held constant for seconds during voltage clamp experiments vs. minutes during changes in exterlow-threshold Ca2+ current through electrical activity, particularly with small changes in external K+. Repetinal K+ concentration in many of the Cai experiments. Second, it is possible that additional Ca2+ influx pathtive action potentials have not been reported in dispersed ways may be activated during strong membrane depolarrat ZG cells despite extensive monitoring of membrane potential (18, 22, 23). In addition, Cai recordings from izations. Other Ca2+ channel types have been identified using voltage clamp techniques in both rat and bovine rat ZG cells are also stable in a control solution including ZG cells (5, 6, 19). Although not seen in the present 4 mM external K’, and Cai rises to a sustained level with elevation of external K+ concentration, consistent with study, these Ca2+ channel currents operate at more positive membrane potentials and typically show less inacthe electrophysiological observations. Second, maintained elevation of a current through the tivation than the low-threshold Ca2’ currents. Inconsistent with this hypothesis, however, are the data using transient, low-threshold Ca2’ channels is also possible, Ca2+ channel inhibitors. Divalent cation inhibitors such despite significant inactivation following initial voltage activation of the Ca2’ current. At membrane potentials as Ni2+ and Cd2+ are equally effective blockers of the Cai where Ca2’ current can be activated and inactivation is elevation stimulated by low and high external K+ conincomplete, a sustained Ca2+ influx should occur (12). In centrations. If different types of Ca2+ channels were ZG cells, these conditions appear to be met for the low- opened across this range of external K+ concentrations, one might expect changes in the sensitivity to these threshold Ca2+ current in a region of membrane potential divalent cation inhibitors. A third possibility is that Na+between -80 and -40 mV, seen as the intersection between current-voltage curves associated with peak cur- Ca2+ exchange could transport Ca2+ into the cell (13) and produce a net Ca2+ influx at membrane voltages more rent and inactivation (Fig. 5B). The magnitude of this sustained current would be small (
DOUGLAS L. TILLOTSON, H. WILLIAMS
Endocrine Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston 02115; and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118 Quinn, Stephen lotson, M. Carter
J., Ulrike Cornwall,
Brauneis, Douglas L. Tiland Gordon H. Williams.
Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosacells.Am. J. Physiol. 262 (Cell Physiol. 31): C598-C606,1992.-Rat and bovine adrenal zona glomerulosa (ZG) cells possessa low-threshold, voltage-dependent Ca2’ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membranepotentials above -80 mV with maximal peak Ca2’ current elicited near -30 mV. Inactivation of the Ca2’ current was half-maximal between -74 and -58 mV, dependingon the external Ca2’ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Ca;) levels.Under basalconditions, Ca; is stable in single rat ZG cells, whereasmore than half of the bovine ZG cells produce repeated Cai transients. These Ca; transients, which are blockedby removal of external Ca2’ or addition of Ni2+,are likely dueto repetitive electrical activity in bovine ZG cells. Ca; responsescan be elicited by small increasesin external K’ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+channels. However, these Cai changes remain robust at high external K’ concentrations (20-40 mM). In experiments combining Ca; measurementsand wholecell voltage clamp,a steepdependence of Cai on membranepotential wasrevealed beginning at depolarizing voltages near a holding membranepotential of -80 mV. A maximal increasein Cai occurred near -30 mV (equivalent to an external K’ concentration of 40 mM), a membrane voltage at which sustainedcurrent through low-threshold Ca2+ channelsshould be negligible. These data raise the possibility of additional voltage-dependentpathways for Ca2+influx. calcium current; adrenal
brane sufficiently
to activate this conductance
(11, 17,
20, 22-24).
Critical to the hypothesis that this Ca2+ conductance is involved in the control of Cai and steroidogenesis is the relationship between membrane potential and the voltage dependency of channel activation and inactivation. Small depolarizations from the resting membrane potential, as observed with millimolar increases in external K’, must significantly activate the Ca2+ conductance. In addition, the channel current must support a sustained increase in Ca2+ influx and accumulation during secretagogue stimulation. Previous studies have shown that elevation of external K+ stimulates an increase in Cai with a dose dependency similar to that for aldosterone production (4, 26). However, there remain questions as to the duration of the Cai change by elevation of external K+ with implications for the kinetics of Ca2+ channel activation and inactivation (1, 10). Moreover, previous studies examining the electrical properties of bovine and rat ZG cells indicate differences between these species that make a comparison of their Ca2+ current(s) and external K+-induced Cai changes important for our understanding of the human adrenal cortex (5, 6, 18, 19, 22, 23). MATERIALS
AND METHODS
IS AN IMPORTANT REGULATOR ofsteroidogenesis in zona glomerulosa (ZG) cells (1, 25). The three major secretagogues, external K+, angiotensin II (ANG II), and adrenocorticotropic hormone (ACTH), require external Ca2’ for aldosterone stimulation and may increase Ca2+ influx to produce a rise in cytosolic-free calcium (Cai) (15, 16, 25-27, 29). While it has been difficult to observe elevation of Cai in ZG cells during ACTH stimulation (16, 29), sufficient data exist to postulate that increases in Ca2+ influx and Cai serve as central elements of the transduction mechanisms of secretagogue stimulation. Recent electrophysiological evidence points to the activation of voltage-dependent Ca2+ channels as a common pathway for Ca2+ entry into ZG cells during stimulation. Membrane voltage and current measurements demonstrate the existence of a Ca2’ conductance in rat and bovine ZG cells (5, 6, 18, 19, 22, 23), and each major secretagogue can depolarize the mem-
Adrenal cellpreparation. Experiments were performed on ZG cellsisolatedfrom rat and bovine adrenal glands.ZG cellsfrom Squague-Dawleyrats were prepared by techniques previously reported (30). Calf adrenalswere obtained from a local slaughterhouse. The adrenalswere sliced with a microtome and decapsulatedusing scissorsand scalpel.The remaining capsular tissuewas placed in a Krebs-Ringer bicarbonate (KRB) solution supplemented with 2% glucose and 4% bovine serum albumin (BSA) and containing crude collagenase(3.7 mg/ml) and deoxyribonuclease(0.05 mg/ml), mincedwith scissorsand incubated for 35 min. The tissue was resuspendedand incubated for an additional 30 min with collagenase(3.7 mg/ml). The supernatant and remaining tissuewere filtered through a 210-micron nylon mesh. After three washesin KRB, the cell suspensionwas placed in a superfusionchamber mounted on the stageof an inverted microscope.The bottom of the chamber consistedof a glasscover slip, which had been coated with a 0.1% solution of commercial gelatin (Knox, Englewood Cliffs, NJ) and treated with a 1 M NaCl solution containing 0.2% concanavalin A (Sigma). The cellsadheredto the glasssurface without apparent adverseeffects to cell viability or membrane properties. The bath chamber was continuously perfusedwith warmed(35-38°C) solution (120mM NaCl, 4 mM KCl, 1.2mM Na2HP04, 1.25 mM CaC12,0.5 mM MgS04, 20 mM HEPES, 0.1% BSA, and 0.2% glucose;pH = 7.4). For experiments combining microspectrofluorimetry and whole cell voltage clamp, cells were plated on glasscover slips
C598
the American
CALCIUM
0363-6143/92
$2.00
Copyright
0 1992
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
cA2+
CHANNELS
AND
CYTOSOLIC
and placed in 60-mm culture dishes. The cells were incubated for 1 to 3 days prior to use. This brief period of culturing makes the formation of gigaohm seals more feasible. The ability of external K+ to stimulate steroid secretion is retained during this time. ELectricaL recordings. The whole cell recording technique was used to voltage clamp the ZG cells (9). Glass pipettes were pulled from Corning 7052 and 8161 capillary tubing with a twostage puller and firepolished to a pipette resistance of 2-10 MQ when filled with internal solution. The pipette was connected to the head stage of a patch-clamp amplifier (List, Darmstadt, FRG), which was mounted on a Huxley manipulator (Custom Medical Research Equipment, Glendora, NJ). Capacitance and series resistance compensation was adjusted to optimize the speed of the voltage clamp. The electrical signals were low-pass filtered at 1.3-3 kHz and displayed on a storage oscilloscope. Signals were digitized and stored with an IBM PC computer for later analysis. Currents were leakage corrected by subtraction of the scaled linear leakage current measured during small hyperpolarizing and depolarizing voltage steps. When using high concentrations of divalent cations, adding of potassium channel blockers, or substituting for Na+, equimolar concentrations of NaCl were replaced. Standard composition of the internal pipette solution for measurement of Ca2+ current was the following (in mM): 140 CsCl, 5 NaCl, 2 MgC12, 1 EGTA, 5 Na2ATP, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES); pH = 7.2. For experiments that investigated total whole cell current, CsCl was replaced by KCl. Measurement of Cai. The measurement of Cai is similar to methods previously described (26, 27). Dispersed ZG cells were incubated for 20 min with fura-2/acetoxymethyl ester (AM) (Molecular Probes) at a concentration of 5 PM in an Eagle’s minimum essential medium (MEM) including 20 mM HEPES, 1.25 mM CaC12, 0.5 mM MgSO,, and 0.1% BSA; pH = 7.47. Cell suspensions were washed and used l-4 h following loading with dye. Test solutions consisted of the standard bath solution and varying concentrations of external K’ with an equimolar adjustment of NaCl. As described previously, the test solutions were applied by puffer superfusion, which allows rapid, local solution changes around an identified ZG cell (24, 26, 27). The whole cell Ca; measurement system consisted of a dual wavelength light source (Photon Technology International), an inverted microscope equipped for ultraviolet fluorimetry (Nikon Diaphot), and a photomultiplier tube (PTI). Furawas used to estimate Ca; by the ratiometric method. Excitation wavelengths were centered at 350 and 380 nm with a bandwidth of 4 nm. Calibration constants were determined by an in vitro method and a dissociation constant (&) of 224 nM was used, as previously reported (8). Estimated Ca; concentrations fell within a range where fura- reliably monitors Cai. Measurement of Ca; during whole cell voltage clamp. Ca; was measured in single ZG cells while under whole cell voltage clamp. A digital video imaging system was used to monitor Ca; as described previously (26). Furawas introduced into the cells via the patch pipette by adding 500 PM of the fluorescent probe to the patch pipette solution containing the following (in mM): 124 KCl, 4 NaCl, 1 Mg-ATP, and 20 HEPES. Fluorescence images were digitized, and the emission signal from an entire cell was integrated and stored as a single value for each excitation wavelength at each time point. This operation was performed in real time at a sampling rate of 2 Hz. Identification of zona glomerulosa cells. Cells were identified as ZG cells based on morphological characteristics described by Tait et al. (28). Cells were spherical with a diameter of 7 to 12 pm. The nucleus was round and occupied much of the cell volume, and the cytoplasm contained numerous small lipid droplets. Data analysis. Ca; responses were analyzed by one-way analysis of variance and the Newman-Keuls multiple comparison
cA2+
IN
ADRENAL
ZG
c599
CELLS
test. All Ca; recordings were digitally Savitsky-Golay smoothing routine.
filtered
with
a U-point
RESULTS
Whole cell current. Rat ZG cells have a dominant K+ conductance at resting and depolarizing membrane voltages (23). Consistent with this finding, a significant outward current is seen during whole cell current recordings under conditions that simulate the physiological ionic composition of the external and internal solutions. Figure IA shows the current response to step changes in voltage from a holding potential of -100 mV. At voltages more positive than -80 mV, both early inward and more slowly developing outward currents were elicited. In Fig. lB, the current-voltage relationship illustrates the voltage dependency of the early and late current. As the membrane voltage was stepped to more positive values, substantial outward current was observed and early net inward current was no longer seen. Potassium channel blockers [Cs’, tetraethylammonium (TEA) and 4-aminopyridine (4-AP)] inhibited the outward current. These results are similar to those we have previously described for outward current in rat ZG cells (3). Inward calcium current. To isolate the inward current from the outward current, K+ was replaced by Cs+ in the internal solution, and, for some experiments, TEA and 4-AP were also added to the bath solution. As seen in Fig. 2A, only inward current was evident following depolarizing voltage steps when the cell was dialyzed with Cs+ internal solution. In bath solution containing 2.5 mM Ca’+, the threshold for activation, the membrane voltage at which active current is first measurable, was between -80 and -70 mV, with maximal peak inward current occurring near -30 mV (Fig. 2B). In other experiments, a decrease in the peak current was found at membrane voltages more positive than 0 mV, but reversal of the inward current was not reached. Substitution of external Na+ by tris( hydroxymethyl)aminomethane (Tris) did not appreciably alter the inward current, implying that Na’ was not significant current carrier. Typically, the inward current in ZG cells was larger in external solutions containing higher Ca2+ concentrations, suggesting that the current was principally carried by Ca2+ (Figs. 2 and 5). Current-voltage relations tended to shift in the depolarizing direction when higher Ca2+ concentrations were used, with the threshold voltage lying between -60 and -50 mV for 10 mM Ca2’. Mn2+ (5 mM; n = 3) or Cd2+ (1 mM; n = 3) blocked the inward current, as has been seen for other Ca2+ currents (data not shown). The inward current displayed strong voltage dependence for activation and inactivation (Fig. 2). The timeto-peak was sharply reduced as the depolarizing voltage step was increased to more positive values. Likewise, substantial inactivation occurred during voltage steps of lOO- to 200-ms duration. Figure 3 shows that the inward current demonstrated similar kinetics and current-voltage relationship when carried by Ba2+. Current inactiuation. Current inactivation was studied by holding membrane voltage at different levels prior to presenting a test voltage step. Figure 4A shows currents elicited in 2.5 and 10 mM Ca2+. The peak current de-
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C600
cA2+
A
CHANNELS
AND CYTOSOLIC
cA2+
B
-SO-'\---
0/ / membrane
-70*-
-80 A
--
-50
voltage, mV -60/ -?-T
'
/
1
0
- 100
-20
-
l
-40 I
d, I
0
-v
- -100
-4oAF----c
] 200 pA
-dj--+---"
ZG CELLS
-200
l
--
-60x/--
IN ADRENAL
Fig. 1. Whole cell current from a single rat ZG cell. A: current responses to voltage steps that are marked to left of each trace. External solution contained 2.5 mM Ca2+. B: current-voltage relationship plotted as peak early current (filled circles) and current at end of voltage step (open circles). Current recordings were leakage corrected. Holding potential, -100 mV. Internal solution contained KC1 as principal ion species.
- -200
creased as the holding potential was varied from -100 mV to more depolarizing values. Peak current was normalized to the peak inward current observed when the membrane voltage was held at -100 mV and was plotted vs. the holding potential in Fig. 4B. Data were fitted to the equation (1 ) h = [l + exp (V - K-J/k]-’
cells. The inward currents of both rat and bovine ZG cells could be isolated from outward currents with Cs+ in the internal solution. Figure 5 compares the inward currents in the presence of 10 mM external Ca2+. The kinetics of the inward current were nearly identical for time-to-peak and the time course of inactivation (Fig. 5A). In Fig. 5B, current-voltage relationships are plotted for both rat and bovine ZG cells. The analysis of current was identical to that described above. To where h represents the inactivation normalized to the inactivation maximal peak current, Vos5is the membrane voltage better compare the current-voltage curves, the peak current at each membrane voltage was normalized to the where h is half-maximal, V is the holding membrane maximal peak current, which was elicited by a voltage voltage, and k is a slope constant. The voltage dependency of this inactivation was steep (k = 4.0) with a Vo.5 step to -20 mV in both examples. Most apparent was of -73 mV for 2.5 mM Ca2’ and -58 mV for 10 mM the similarity of the current-voltage relationship (Fig. 5B), indicating the same voltage dependency for the Ca2+. Comparison of calcium current in bovine and rat ZG inward Ca2+ current in both species.
B
A 2.5 mM Ca2+ -70
membrane -80
-60
-40
potential,
-20
mV
20
0
-iv
-60 -40
'200
-20* 0 Y
1
v
1
100 msec
1
2 g 2 4 gct
Fig. 2. Inward current from rat ZG cell. A: traces represent inward current elicited by voltage steps in 2.5 mM Ca2’. Voltage steps are noted to left of each record. B: current-voltage curve for peak inward current in 2.5 mM Ca2’. Current traces were leakage corrected. Holding potential, -100 mV.
;b *
t-
J 125 pA
J
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CHANNELS
cA2+
AND
CYTOSOLIC
cA2+
IN
ADRENAL
ZG
CELLS
C601
A membrane
5 mM Ba2’
-60
-40
potential,
mV
-20
0
20
f
B
200 5 z
O/$
50 msec
Fig. 3. Inward current carried by barium. A: traces represent inward current elicited by voltage steps in 5 mM Ba2’. Voltage steps are noted to left of each record. B: current-voltage curve for peak inward current in 5 mM Ba2’. Current traces were leakage corrected. Holding potential, -80 mV.
4
mM and diminished substantially with 40 mM to a level roughly equivalent to the Cai response at 10 mM external K+. The mean amplitude change at each external K+ concentration was larger for the rat than the bovine, consistent with the larger Ca2+ current found in rat ZG cells. The increase in Cai stimulated by 10, 20, and 40 mM external K+ was completely blocked by 1 mM Ni2+ or 1 mM Cd2+ (data not shown). Lower concentrations of these divalent cations produced partial inhibition. These results are similar to those we have previously described for Cai responses to smaller elevations of external K+ (26) Membrane voltage and Cai changes. Increases in exter‘cai responsesto external K+ in rat and bovine ZG cells. As observed in previous studies, Cai levels in ZG cells nal K+ concentration can depolarize the membrane powere exquisitely sensitive to changes in external K+ tential to a new value, analogous to the voltage-clamp concentration. Elevation of external K+ from a basal technique. In rat ZG cells, elevation of external K+ value of 4 mM to concentrations ranging from 6 to 40 concentration by the puffer pipette method can produce that is complete within 100 mM produced prompt increases in Cai (Fig. 7). At low a membrane depolarization concentrations of external K+, the Cai change was sus- ms and sustained for the duration of stimulation (data tained with little change in amplitude in rat ZG cells; not shown). Voltage clamp offers even faster and more precise control of membrane voltage and eliminates any however, Cai transients were often found under similar conditions in bovine ZG cells. Of the bovine ZG cells additional effects resulting from the elevation of external tested with stimulation by 6 mM external K+, over 50% K + To directly evaluate the relationship between memdisplayed rapid, repeated Cai transients that had a greater frequency and/or amplitude than under basal brane voltage and Cai, techniques were combined to monitor Cai during a series of voltage steps while the conditions of 4 mM K+. At high external K+ concentrations, the Cai change was typically stable in rat and individual ZG cell was placed under whole cell voltage bovine cells, although a slow decline from maximal Cai clamp (Fig. 9). As the magnitude of the depolarizing levels was observed in some cases. More than 90% of the voltage step was increased, the change in Cai also inbovine and rat ZG cells displayed a Cai response when creased with a threshold voltage between -80 and -70 subjected to high external K+ concentrations. mV to a maximal response at -31.43 t 4.25 mV (n = 8). The dose-response relationships (Fig. 8) for Cai re- Further depolarization progressively reduced Cai values, sponses stimulated by external K+ indicate that the resulting in a 44 t 15% (n = 8) decrease in the emission amplitude of the Cai change rises steeply between 4 and ratio change with voltage steps to values between 0 and 10 mM external K+ in both rat and bovine ZG cells. At +4O mV. Longer voltage steps to voltages that produced higher external K+ concentrations, the Cai peaked at 20 near-maximal Cai changes demonstrated a sustained Cai Basal Cai levels in rat and bovine glomerulosa
cells. Cai
was monitored in single ZG cells isolated from rat and bovine adrenals. The mean basal Cai was similar for rat, 98 t 4 (SE) nM (n = 258), and bovine, 113 t 9 nM (n = 45), ZG cells. Basal Cai was defined as the level between oscillations. Basal Cai was stable in rat ZG cells, but over 50% of bovine cells displayed spontaneous Cai transients (Fig. 6). When these Cai oscillations were present in bovine ZG cells, they tended to have a consistent amplitude for each individual cell. These basal Cai transients were completely inhibited by ion substitution of Mg+ for external Ca2+ or by the addition of 1 mM Ni2+ (Fig. 6)
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
C602
CA2+ CHANNELS
AND
A 2.5 mM Ca2'
+
-
-60-J-0----
-40-h
-7oiL_-
-5o&.#-----
ZG
CELLS
Cd2+, and Ni2+ substantially block it. Threshold voltage for activation lies between -80 and -70 mV in superfusates containing Ca2+ concentrations (254.0 mM) close to the physiological range. Significant inactivation is observed during sustained depolarizations, which is largely dependent on voltage rather than Ca2+ entry, because the kinetics of inactivation are observed in the presence of a strong Ca2+ chelator [ 1 mM ethylene glycolbis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA)] in the patch pipette and are retained when
mM Ca2'
10
cA2+ IN ADRENAL
CYTOSOLIC
-
A rat I pre-pot. iI1
msec ‘J
100
5
bovine
10 mM Ca”
10 mM Ca”
bb,bb
10. 08. 06. s
8
\
04. .
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OL. c m C I -110
I
I
I
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\
I
\\ \ \
I
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-70
pre-potential,
\\
I
L.
100 msec
B \
\ c \ 1
1.0 ‘La I
0.8
-50
mV
rat ZG cells. A: currents elicited by Ca2’ and -30 mV for 10 mM Ca2’ (pre-pot) shown to left of each trace. for 2.5 mM Ca2’ and 5 s for 10 mM Ca2+. Data were from 2 different rat ZG cells. B: voltage dependency of inactivation. Peak inward currents for a voltage step to -40 mV were normalized to peak current value elicited when holding potential was -100 mV (ha) and plotted vs. pre-potential value. Smooth curves through data points were derived from &. I with the Vo.5 of -73 mV for 2.5 mM Ca2+ (circles) and -58 mV for 10 mM Ca2’ (squares) and k value for 4.0.
plateau achieved within the first 5 s and lasting for the pulse duration of 20 s (data not shown). Simultaneous measurements of inward Ca2’ current and Cai were not possible as the outward current was not inhibited. An external Ca2+ concentration of 1.25 mM was used to approximate physiological conditions. DISCUSSION
voltage-dependent
0 0 4
100 msec
Fig. 4. Current inactivation from a voltage step to -40 for 2.5 mM from different holding potentials Pre-potentials were held for 10 s
A similar
4 a
0” Lo
Ca2+ current is found in
ZG cells of rat and bovine adrenal tissue that appears
responsible for increases in Cai stimulated by small physiological elevations in external K+ concentration. The membrane conductance change giving rise to this current is selective for divalent cations. Both Ca2’ and Ba2’ are carried through this conductance pathway, while Mn2+,
s
i
8
0 0I . \ \
! ! !
\
0.4 t
‘0”
\
‘\‘\II -7 ‘\ 2i 0.6
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I
0.2 .
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0.2
11
-90
11
11
-70
-50
membrane
II
11
-30
-10
potential,
mV
J
0
10
Fig. 5. Comparison of rat and bovine zona glomerulosa cells. A: traces represent inward current elicited in 10 mM Ca2+ by voltage steps, indicated to left of each record. Note that current calibrations are different for each ZG cell. Current records were leakage corrected and holding potential was -80 mV. B: current activation: peak inward current was normalized to maximal value elicited with a voltage step to -20 mV and plotted on right-hand side of graph (rat: open squares; bovine: open circles). Maximal peak current was 750 and 125 pA for rat and bovine ZG cells, respectively. Current traces are shown in previous figure. Current inactivation: peak inward currents for a voltage step to -30 mV from different holding pre-potentials were normalized to maximal value elicited with a holding potential of -100 mV and plotted on left-hand side of graph (rat: filled squares; bovine: filled circles) vs. pre-potential value. Smooth curves through data points were derived from Eq. 1 with Vo.5 = -58 mV and k = 4.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
CHANNELS
CA2+
CYTOSOLIC
“0” Cd +
r
4oor
4OOr
c
..-
IN
cA2+
bovine
bovine
2
AND
i t
1 mM
Ni2+
ADRENAL
ZG
100 400
,“bb’nwf .ff
.
Fig. 6. Basal cytosolic calcium (Ca;) and effects of nominal zero calcium and nickel in bovine zona glomerulosa cells. Shown are representative basal recordings from 4 individual bovine ZG cells. Roughly half of bovine ZG cells exhibited basal Cai transients. Timing of external calcium removal or addition of 1 mM nickel is indicated by arrows. External Ca2’ concentration, 1.25 mM.
M&p%%&
44
100
100
I
I 1 minute
inactivation are more negative than in many other reports of low-threshold Ca2+ currents, which is possibly due to the high divalent cation concentrations typically employed when evaluating Ca2+ currents. Another interesting feature of the inward current of ZG cells is the predominance of low-threshold Ca2’ current over other types of Ca2+ current. In most other cell types, the highthreshold, L-type Ca2+ current is much larger than the low-threshold Ca2’ current. The magnitude of the lowthreshold Ca2’ current may, in part; be responsible for the unusual sensitivity of ZG cells to external K+ concentration, where very small membrane depolarizations
external Ca2+ is replaced by Ba2+. Our experimental conditions did not allow for the direct evaluation of Ca2+dependent inactivation. The Ca2’ current available for activation is dependent on the membrane potential. Half-maximal current inactivation was observed between -73 and -58 mV, with the most negative values found at external Ca2’ concentrations closeto the physiological range, as reported in a previous study using rat ZG cells (18). Therefore, the Ca2’ current found in ZG cells resembles the low-threshold, T-type Ca2+ current characterized in many other tissues (2, 7, 31). In our experiments, the voltage dependences of activation and of rat
C603
1
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Er -5 100 3 2 400 52 2 3
CELLS
bovine external
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10 20 mM Fig. 7. Time course of Cai response to different external potassium concentrations in rat and bovine zona glomerulosa cells. Shown are representative Cai recordings from 4 different rat and bovine ZG cells during puffer superfusion with elevated K+. Bar above the Cai recordings indicates onset and termination of external K+ elevation. Concentration of external K+ is indicated for each pair of traces. External Ca2’ concentration, 1.25 mM.
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Fig. 8. Relationship between maximal Cai change and external potassium concentration. Maximal Cai change is increase above basal levels when external K+ concentration is elevated from a basal value of 4 mM. Each data point represents mean t SE from at least 10 individual rat or bovine ZG cells at each external K’ concentration.
can lead to substantial increases in Ca2’ influx, Cai, and hormone secretion. The Cai current described in this study is consistent with our previous voltage recordings of Ca2+ action potentials characterized in rat ZG cells (23). The thresholds for activation and current inactivation have similar voltage dependency, and the selectivity and blockage of the underlying conductance are the same. Other recent studies using voltage-clamp techniques have also demonstrated Ca2’ currents in rat and bovine ZG cells. Matsunaga et al. (18) found only a transient Ca2+ current with a low-threshold for activation in freshly dispersed rat ZG cells, while other investigators have reported as many as three different Ca2’ currents in cultured rat ZG cells (6, 22) with voltage dependencies similar to the T-, L-, and N-type calcium channels of the chick sensory neuron (21). The reason for differences among these studies is unknown but may involve the use of freshly dispersed vs. cultured ZG cells. Studies using bovine ZG cells have consistently found two types of Ca2+ current (5,19). The low-threshold current had properties similar to that described in our study and was found in all bovine ZG cells, while a high-threshold Ca2+ current similar to the L-type was reported to be present in a small percentage of the cells (5). Adrenal ZG cells also possessvoltage-dependent outward currents that may be important in the stimulation of aldosterone production by its major secretagogues (3). The outward current is likely to be carried by K+ because it follows the ionic gradient for K+ across the plasma membrane and is blocked by replacing K+ by Cs+ in the internal solution. Other studies have also demonstrated K+ conductances in ZG cells both at resting membrane potential and with membrane depolarization (3, 22, 23). The major secretagogues induce membrane depolarizations either by reducing the driving force for K+ (elevation of external K’) or by blocking one or more of these K+ conductances (ANG II and ACTH) (3, 20, 22-24). This membrane depolarization may be a common transduction mechanism for the stimulation of Ca2+ influx
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Fig. 9. Relationship between membrane voltage and Cai concentration. A: top trace shows change in ratio of light emission stimulated by excitation wavelengths of 350 and 380 nM, respectively. Bottom trace indicates timing and amplitude of a 2-s voltage step applied from a holding potential of -80 mV to a bovine ZG cell under whole cell voltage clamp. Interval between voltage steps varied to allow return to basal Cai values. B: plot of maximal change in emission ratio vs. membrane voltage. Change in emission was corrected for rundown of Cai response during course of experiment by comparing Cai responses to equivalent voltage steps to -20 mV applied at different times during experiment and assuming a linear decay of Cai response over time. Similar results were found for bovine (n = 7) and rat (n = 1) ZG cells. External Ca2’ concentration, 1.25 mM.
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produce significant changes in Cai. Previous studies of through the opening of voltage-dependent Ca2+ channels. Thus the low-threshold Ca2+ current is likely to play an membrane voltage and Cai responses in rat ZG cells are important role in the regulation of Cai levels and aldosteconsistent with the hypothesis of a sustained Ca2+ current through low-threshold channels. rone production for a number of its secretagogues. From our previous work, rat ZG cells were found to Large changes in external K+ concentration also prohave an average resting membrane potential of -78 mV duce sustained Cai responses in bovine ZG cells. Under at an external K+ concentration of 4 mM. The relationthese conditions, bovine ZG cells may also utilize a ship between membrane voltage and external K+ was 45 sustained calcium conductance to produce stable Cai signals. This is substantiated with Cai measurements mV voltage per lo-fold change in external K+ concentration (23). Because the threshold voltage for Ca2+ channel under voltage-clamp conditions when voltage steps of 5activation and the membrane voltage for half-maximal s duration produce a continuously increasing Cai response likely due to a sustained Ca2+ current. Moreover, inactivation lie close to the resting membrane potential, sustained current through these Ca2+ channels should voltage pulses of 20-s duration elicited sustained Cai only occur with small membrane depolarizations such as changes that rapidly returned to basal levels with the the 7-mV depolarization predicted for an external K+ termination of the voltage step. Had Ca2+ current completely inactivated, a decline of Cai would have been elevation from 4 to 6 mM. This hypothesis is consistent expected during a sustained pulse. These Cai data can be with the steep dependence of Cai level and aldosterone production on external K+ concentration between 4 and contrasted with Ca2+ current recordings, which show 10 mM (5-7). Likewise, experiments using voltage pulses near complete inactivation by the end of a 200-ms voltage significant in- step, indicating the sensitivity of Cai as an indicator of to activate Ca2+ current demonstrate creases in Cai with voltage steps from -80 to -70 mV. maintained Ca2+ influx. Stimulation by external K+ or direct membrane voltage Some features of Cai responses during stimulation with high K+ concentrations or large positive voltage steps change elicits equivalent Cai responses, pointing to voltage as the primary transduction element for external K+. cannot be easily explained by a sustained conductance through low-threshold Ca2+ channels. At membrane voltOther effects of increasing the concentration of external K+ probably do not contribute to the Cai or aldosterone ages more positive than -50 mV (external K+ concentrations greater than 20 mM), sustained Ca2+ current responses. Questions remain as to the mechanism for sustained through these channels should be vanishingly small, yet secretagogue-induced Ca2+ influx and the sustained rise the Cai responses reach their peak amplitudes in this voltage and external K+ range. Two possible explanaof Cai observed with K+ stimulation in rat and bovine cells. The present findings suggest two possible ways by tions for these disparate results are the following. First, which Ca2+ influx may remain elevated during long-term analysis of low-threshold Ca2’ current may not accusecretagogue stimulation. First, the transient Ca2+ cur- rately describe the sustained current through this conrent may be repeatedly activated by membrane depolarductance system. There may be inaccurate determinaization resulting from electrical activity. Trains of spike tions of the voltage dependency of the sustained Ca2’ activity have been observed in some cells from kitten current because the current amplitudes are very small in adrenal capsular tissue at rest and during K+- or ANG the voltage range of interest and external Ca2+ concenrange were used in II-stimulated depolarization (20). Information on elec- trations greater than physiological trical activity of bovine ZG cells is not available. How- the electrophysiological measurement of the Ca2+ curever, bovine ZG cells exhibit repeated Cai transients rents. Alternatively, the voltage dependency of current inactivation may be significantly different when memduring basal and membrane depolarized conditions. Thus bovine ZG cells may repeatedly activate the transient, brane voltage is held constant for seconds during voltage clamp experiments vs. minutes during changes in exterlow-threshold Ca2+ current through electrical activity, particularly with small changes in external K+. Repetinal K+ concentration in many of the Cai experiments. Second, it is possible that additional Ca2+ influx pathtive action potentials have not been reported in dispersed ways may be activated during strong membrane depolarrat ZG cells despite extensive monitoring of membrane potential (18, 22, 23). In addition, Cai recordings from izations. Other Ca2+ channel types have been identified using voltage clamp techniques in both rat and bovine rat ZG cells are also stable in a control solution including ZG cells (5, 6, 19). Although not seen in the present 4 mM external K’, and Cai rises to a sustained level with elevation of external K+ concentration, consistent with study, these Ca2+ channel currents operate at more positive membrane potentials and typically show less inacthe electrophysiological observations. Second, maintained elevation of a current through the tivation than the low-threshold Ca2’ currents. Inconsistent with this hypothesis, however, are the data using transient, low-threshold Ca2’ channels is also possible, Ca2+ channel inhibitors. Divalent cation inhibitors such despite significant inactivation following initial voltage activation of the Ca2’ current. At membrane potentials as Ni2+ and Cd2+ are equally effective blockers of the Cai where Ca2’ current can be activated and inactivation is elevation stimulated by low and high external K+ conincomplete, a sustained Ca2+ influx should occur (12). In centrations. If different types of Ca2+ channels were ZG cells, these conditions appear to be met for the low- opened across this range of external K+ concentrations, one might expect changes in the sensitivity to these threshold Ca2+ current in a region of membrane potential divalent cation inhibitors. A third possibility is that Na+between -80 and -40 mV, seen as the intersection between current-voltage curves associated with peak cur- Ca2+ exchange could transport Ca2+ into the cell (13) and produce a net Ca2+ influx at membrane voltages more rent and inactivation (Fig. 5B). The magnitude of this sustained current would be small (
