0013.7227/92/1311-0408$03.00/0 Endocrinology Copyright D 1992 by The Endocrine
Vol. 131, No. 1 Printed m Ii S A
Society
Angiotensin II Receptor-Mediated Calcium Bovine Adrenal Glomerulosa Cells* CLARA
AMBROZ
Endocrinology Development,
AND
KEVIN
and Reproduction National Institutes
Influx
in
J. CATT Research Branch, National Institute of Child Health of Health, Bethesda, Maryland 20892
ABSTRACT The cytoplasmic calcium ([Ca”],) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca’+ mobilization, followed by a sustained plateau phase that is dependent on Ca” entry from the extracellular fluid. In Furaloaded cells, the calcium channel antagonists, nifedipine (1 pM) and verapamil (20 PM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca”], when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca’+],. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca’+ channel blocker, LaCl,, (10 @M), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca” signal was not affected by treatment with pertussis
and Human
toxin (100-300 rig/ml for 12 h). The prior addition of specific AIIantagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sarl,ThrR]AII, [Sar’,Ala”]AII, and [Sar’,Ile”]AII) completely inhibited the AII-induced Ca’+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 set to 5 min after AI1 caused progressively less attenuation of the sustained Ca”+ signal. After 5 min, addition of antagonists did not inhibit the agonistinduced Ca’+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca’+], could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca’+ influx despite receutor blockade. It is nossible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca’+], response. (Endocrinology 131: 408414,1992)
A
NGIOTENSIN II (AII), a major physiological regulator of aldosterone production by adrenal glomerulosa cells, stimulates steroidogenesis and hormone secretion by activation of phosphoinositide hydrolysis and elevation of cytoplasmic Ca2+ concentration ([Ca’+],) (l-7). Although the role of Ca2’ as an intracellular messenger for the action of AI1 is well established, the mechanisms responsible for the agonist-induced increase in [Ca’+], are not yet clear. The secretory response to agonist stimulation in adrenal glomerulosa cells is accompanied by an initial transient rise in [Ca’+], that is largely derived from mobilization of intracellular Ca2+ stores, and is followed by a prolonged plateau phase that depends on Ca2+ entry across the plasma membrane. While the initial transient increase in [Ca’+], is coincident with and attributable to the early peak of Ins(1,4,5)P, production (6, 8, 9), the entry pathway(s) that provide Ca2+ to sustain the plateau phase of the AII-induced [Ca’+], response are not well defined (4, 6, 7, 10, 11). There are species differences in the extent to which voltage-sensitive calcium channels (VSCC) participate in AIIinduced [Ca2+li responses and aldosterone production in rat and bovine glomerulosa cells. These are exemplified by the
ability of the dihydropyridine calcium channel agonist, BAY K 8644 (BK 8644), to enhance cytoplasmic Ca2+ and aldosterone secretion in AII-stimulated rat but not in bovine glomerulosa cells (12, 13). Several reports have shown that AIIinduced aldosterone production is reduced by antagonists of VSCC in both species (1, 2, 14-16), but the enhanced Ca2+ entry observed in AII-stimulated bovine glomerulosa cells is less affected by such compounds (2, 13). Recent evidence has indicated that guanine nucleotide regulatory (G) proteins are involved in the regulation of Ca2+ channel function in several cell types (17-21), and it has been suggested that AII-mediated Ca2+ influx is dependent on the action of a regulatory G-protein (22). In addition, receptor-operated Ca2+ channels have been proposed to mediate Ca2+ entry through pathways other than L-type Ca2+ channels; however, there is relatively little evidence to support the existence of such channels (23). In the present study, we examined the potential mechanisms of AII-mediated Ca2+ entry in bovine glomerulosa cells to better define their participation in the maintenance of agonist-stimulated [Ca’+] signaling responses. Materials
Received December 19, 1991. Address correspondence and requests for reprints to: Dr. Kevin J. Catt, Room Bl-L400, Building 10, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marvland 20892. *This investigation was supported by The National Institute of Child Health and Human Develoument, National Research Service Award HD-07383-03, from the EAdocrinology and Reproduction Research Branch.
and Methods
Materials Cell culture media were prepared by the NIH Media Supply Unit (Bethesda, MD). AII, [Sar’,Th;‘]AII, [Sar’;IleR]AII, and [Sar’,Ala’jAiI were purchased from Peninsula Laboratories, Inc. (San Carlos, CA). The nonpeptide antagonist, DuP 753, was kindly provided by Dr.‘P. C. Wong (DuPont, Wilmington, DE). [a?‘]NAD (5 mCi/ml) was obtained from DuPont-New England Nuclear (Boston, MA). lZ51-AII was obtained from Hazleton Labs (Vienna, VA), and 125-I aldosterone RIA kits from
408
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CALCIUM
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Diagnostic Product Corp. (Los Angeles, CA). Fura-2/AM was obtained from Molecular Probes, Inc. (Eugene, OR). All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO).
Preparation
of bovine glomerulosa
cells
Bovine adrenal glomerulosa cells were prepared and cultured as described (24) with the exception that metyrapone (5 PM) was included in the culture medium. Cells were plated in 6-well culture dishes at a density of 1 million cells per ml for cytoplasmic Ca’+ measurements and in 24-well plates at a density of 0.5-l million per well for steroid production assays, and were maintained in culture for 2 or 3 days. One day before Furaloading or aldosterone production experiments, the culture medium was changed to a serum-free solution. For cytoplasmic Ca*+ assays, the cells were collected and incubated with Fura-2/AM (final concentration: 1 PM) as described previously (25).
Measurement
of cytoplasmic
calcium
After incubation with Fura-2/AM the cells were washed twice in a modified Krebs-Ringer solution (KRB) (1.8 rnM Ca&, 2.4 mM KCl, 123 rnM NaCl, 0.8 mM MgC12, 1.2 mM KH2POI, 5 mM NaHC03, and 10 mM glucose) supplemented with 20 mM sodium HEPES (pH 7.4), resuspended in the same buffer at a concentration of 2-4 million cells per 100 ~1 and kept at room temperature until analyzed for [Ca”+], responses. For each [Ca’+], determination a loo-p1 aliquot of washed Fura-2-loaded cells was transferred to a quartz cuvette containing 1.9 ml modified KRB. Analysis of [Ca”+li responses in the absence of extracellular Ca*+ was performed by resuspension of the cells in a cuvette containing KRB without CaClz immediately before spectrofluorimetric analysis. All fluorescence determinations were performed in a PTI-Deltascan spectrofluorometer (Photon Technology International Inc., South Brunswick, NJ) connected to a microcomputer for data storage and analysis. The cuvette holder was equipped with a magnetic stirrer and maintained at a constant temperature of 30 C. Data were collected using dual wavelength excitation at 340 and 380 nm and an emission wavelength of 500 nm (slit width was 4 nm for excitation and emission). Cytoplasmic Ca” concentrations were calculated from the fluorescence ratio according to the method of Grynkiewicz et al. (26) and the values were corrected for cell autofluorescence using software provided with the PTI-Deltascan system.
Aldosterone
production
Before aldosterone production measurements, cultured bovine adrenal glomerulosa cells were washed twice and incubated for 1 h in bicarbonate-buffered modified M-199 at 37 C in an atmosphere of 5% C02/95% O2 to remove metyrapone. At the end of this period the medium was replaced and the cells were incubated with selected concentrations of reagents for 15 min to 2 h at 37 C. Cells were then placed on ice, and the medium was collected and stored at -20 C for aldosterone analysis by RIA.
GLOMERULOSA
Role of dihydropyridine-sensitive induced [Ca2+], responses
ADP-Ribosylation studies were performed according to a previously described protocol (27). Briefly, glomerulosa membranes (20-40 pg) from control cells and pertussis toxin (PT)-treated cells were added to tube containing 0.1 ml 100 rnM Tris (pH 8.0), 10 rnM thymidine, 1 mM ATP, 100 KM GTI’, 2.5 mM MgCl,, 1 mM EDTA, 10 mM dithiothreitol, 2 FCi [a-?‘]NAD, and l-2 fig activated PT. Samples were incubated for 3060 min at 37 C, and the reaction was terminated by addition of 200 ~1 gel sample buffer containing 4% (wt/vol) sodium dodecyl sulfate (SDS), followed by boiling for 5 min. Proteins were separated by SDS-polyacrylamide gel (12%) electrophoresis, followed by autoradiography.
Statistical
analysis
Student’s t test was used to determine between means.
the significance
of differences
calcium channels
in AII-
As illustrated in Fig. 1, the rise in [Ca’+]i induced by 100 nM AI1 was biphasic, with a transient peak that was only slightly reduced in the absence of extracellular Ca*+, followed by a sustained phase of elevated [Ca’+], that was completely abolished in Ca2+-free medium. The addition of 20 PM verapamil to cells before stimulation by 2 nM AII, or during the sustained phase of the AII-evoked Ca2+ response, had no consistent effect on the peak height but caused a partial decrease of the plateau phase (Fig. 2A). Preincubation of the cells with 1 PM nifedipine, or addition of the VSCC antagonist during the sustained phase of the AII-induced [Ca*+]i response, had the same partial inhibitory effect as verapamil (Fig. 2B). The addition of 50-100 nM BK 8644 to bovine adrenal glomerulosa cells caused a slow and minor increase in basal [Ca’+J, but did not enhance the subsequent Ca*+ response evoked by 2 nM AII. Addition of the dihydropyridine agonist during the plateau phase caused a small and additive increase in the Ca*+ signal (Fig. 2C). These findings, which were confirmed in five different experiments, were consistent with previous observations (2, 12) that the majority of the rise in cytoplasmic Ca 2+ induced by AI1 in bovine adrenal glomerulosa cells is independent of VSCC activation. In 2-h incubations, nifedipine (1 FM) inhibited the aldosterone production induced by AI1 (1 nM) by 30%, from 1186 + 156 to 832 + 85 pg/ml (means f SD, n = 4, P < 0.05) (data not shown). Effects of inorganic
calcium channel
blockade
The ability of the inorganic calcium channel blocker, LaC&, to reduce or abolish the agonist-induced Ca*+ entry process was evaluated in AH-stimulated glomerulosa cells. The results obtained from 10 determinations performed in 3 different cell preparations are summarized in Fig. 3. Addition of 600 r
C
studies
409
Results
5 400 ADP-Ribosylation
CELLS
,-GN+ ci 200
Cont & Ca2 ’ -free
-D
I 0
All 1100 nM1
100
200 Time
300
(set)
Fro. 1. Extracellular Ca’+ dependence of the AII-induced sustained [Ca”+], response. Fura 2-loaded cells were centrifuged and resuspended in Ca”+-free medium immediately before the beginning of the recording (time 0), and 100 nM AI1 was added to the cuvette within 2 min. The data are typical traces from experiments repeated more than six times.
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CALCIUM
t
I
0 -
INFLUX
I
I
I
IN AII-STIMULATED
L
0
,I
I
I
200 Time (set)
‘Lo
I
I
300
400
FIG. 3. Inhibition of the sustained phase of the AII-stimulated [Ca”], response by LaC13. The inorganic Ca2+ channel blocker (10 PM) was added before (truce a) or during the plateau phase (truce b) of [Ca”li responses stimulated by 2 nM AII. The traces shown are representative of 10 determinations made in 3 different experiments.
Nif.
I
1992 No 1
La3+ I
0’
1
Endo. Vol131.
All
La3 ’
mrB
‘(%-G-i
CELLS
I
100200300400500600 Time (set)
I
GLOMERULOSA
1
100200300400500600 Time (set)
400 5
r Normal
Medium
300
c F 200 N m o
100 t
I
0 Time
(set)
FIG. 2. Effect of organic Ca2+ channel antagonist and agonist compounds on AII-stimulated cells. A, Verapamil (20 FM) was added at time 0 (truce a) or during the sustained phase of the Ca*+ response to 2 nM AI1 (trace b); B, 1 PM nifedipine was added before (trace a) or after (truce b) the addition of 2 nM AI1 at the times indicated. This concentration of nifedipine caused a slight depression of the baseline in one of four cell preparations studied. C, BK 8644 (BK) (100 nM) was added when indicated by the arrow to cells subsequently stimulated by 2 nM AI1 (truce a) or to cells previously stimulated with 2 nM AI1 (truce 6). Similar results were obtained with a lower dose of BK 8644 (50 nM). The traces shown in the panels represent the results of three (A), four (B) or five (C) different experiments.
10 PM LaC13, either before or after stimulation of the cells by 2 nM AII, did not affect the initial peak response but reduced the sustained phase of the [Ca’+], response by up to 50%. Higher concentrations of LaC4 (up to 20 PM) abolished up to 80% of the AII-induced plateau phase of the calcium response. However, such doses also caused depression of the basal [Ca’+]i level in unstimulated cells (data not shown). In 2-h incubations, LaCL (10 PM) caused a significant (P C 0.01) reduction in AII-stimulated aldosterone production, which decreased from 166 + 23 pg/ml in control cells to 55 & 20 pg/ml (mean f SD, n = 3, P < 0.01). Role of G-proteins
in AII-induced
calcium entry
To evaluate the role of Gi or G, proteins in the mechanism of agonist-stimulated calcium entry in bovine adrenal glomerulosa cells, we examined the effect of pertussis toxin (PTX) on the calcium response.Cells pretreated for 12 h with
400
I
100 Time
Ca*+
- Free
I
200 (set)
300
I
I
Medium
r
I
I
200 300 100 Time (set) FIG. 4. Cytoplasmic calcium responses to AI1 in PTX-treated glomerulosa cells. Upperpanel, Representative tracings of the [Ca”+], response to 2 nM AI1 in cells pretreated with PTX (300 mg/ml for 12 h us. control cells. Lower doses of PTX (100 rig/ml for 12 h) had similar effects. Lower panel, Experimental conditions were the same as in the upper panel with the exception that the cells were centrifuged and resuspended in Ca*+-free medium at the beginning of the recording. These results (upper and lower panel) summarize observations from six different experiments. 0
100-300 rig/ml PTX showed no change in the Ca2+profile evoked by 2 nM AI1 (Fig. 4, top). We sometimesobserved a slight reduction of the [Ca’+], peak which appeared to be dependent on the presence of extracellular Ca2+ (Fig. 4, bottom). However, this effect was present only in two of the six experiments performed with PTX. Likewise, in accordance with previous reports (27), no inhibitory effect on aldosterone production was observed (data not shown). The effectiveness of the PTX treatment was confirmed by SDS-polyacrylamide
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CALCIUM
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IN AII-STIMULATED
GLOMERULOSA
mr
gel electrophoresis of solubilized membrane proteins prepared from cells preincubated with the lowest PTX concentration used (100 @ml). Autoradiograms of such gels revealed that ADP-ribosylation of a 41-kilodalton protein corresponding to the a-subunit of Gi and G, was reduced by about 95% in membranes from PTX-treated cells vs. control cells (Fig. 5).
DuP 753 (50 FM) Added
t
Effects of receptor antagonists
on All-stimulated
calcium entry
Evidence for the participation of agonist-activated Ca2+ channels in the maintenance of AII-induced increases in [Ca’+], was sought by exposing AII-stimulated glomerulosa cellsto specific AI1 antagonists at selectedtimes after addition of the agonist. As shown in Fig. 6, both DuP 753 and [Sar’, Ile*]AII caused marked inhibition of the plateau phase of the Ca2+ signal when added soon after 2 nM AII. The initial [Ca’+]i spike was maintained even when the time interval between the administration of the agonist and antagonist was only 10 set, but the subsequent plateau phase was abolished or attenuated. Addition of the antagonists at later time points caused progressively less inhibition of the sustained phase of Ca2+entry, and had no effect on the [Ca’+], plateau when performed more than 5 min after stimulation with AII. The concomitant administration of agonist and antagonist, as expected, caused complete inhibition of the AII-stimulated Ca2+signal (not shown). The data shown in Fig. 7 represent the plateau [Ca’+], levels reached after addition of DuP 753 and three peptide antagonists, all of which gave similar results. They are expressed as the mean + SEM of at least three different measurementsat each time point, and demonstrate the progressive lossof inhibition of the AIIinduced [Ca2’J response by delayed addition of the AI1 antagonists, even in concentrations up to 25,000-fold molar excess. Discussion It is well established that AI1 and extracellular potassium (K+), two primary regulators of aldosterone production by adrenal zona glomerulosa cells, exert their stimulatory effects through an increase in cytoplasmic Ca2+ concentration (6, recent review). Although the Ca2+ entry through which K+ evokes aldosterone production is clearly dependent on acti-
123456
36-, FIG. 5. ADP-Ribosylation of membrane fractions from control and PTX-treated bovine adrenal glomerulosa cells. Pretreatment of 3-day glomerulosa cell cultures with PTX (100 rig/ml for 12 h) inhibited subsequent in vitro ADP-ribosylation of a M, = 41,000 protein. Membrane fractions were prepared from control cultures (lanes 1, 3, 5) and PTX-treated cultures (lanes 2, 4, 6); the same cell preparations were used for the analysis of AII-induced [Caz+li responses.
411
CELLS
I
0
300
=
r I I
[Sar’,
lle*lA11
AI1 (2 nM) +
I
loo
I
200 300 Time (set)
at: (set)
I
400
(I FM)
I
I
I
I
200 300 400 Time (sec) FIG. 6. Inhibitory effects of AI1 antagonists on agonist-induced lCa*‘l, responses. Upper panel, Progressive loss of the inhibitory effect of Dup 753 (50 pM) added at increasing intervals after 2 nM AII; lower wanel. similar loss of inhibition bv 1 uM lSar’.Ile”lAII added at increas: mg intervals after 2 nM AD. The times of addition of antagonist are indicated by the arrows, and at the side of each record. The control traces were identical with the 300-set traces and are not shown. The data are representative of results obtained in nine experiments. 0
loo
vation of VSCC, the mechanism(s)responsible for AII-stimulated Ca2+movement acrossthe cell membrane are not yet understood (2, 3, 7, 10, 13, 14, 28). There is ample evidence that the rise in [Ca2+]i which follows activation of the AI1 receptor is caused by at least two distinct processes:a transient release of Ca2+ into the cytoplasm from intracellular stores, and an accompanying sustained increase of Ca2+ influx across the cell membrane. The rapid and transient [Ca2+]iresponseis known to be mediated by phosphoinositide hydrolysis and increased production of Ins(1,4,5)P3 (6), but relatively little is known about the Ca2+ entry process. Recent studies using patch clamp techniques have indicated the presence of two distinct types of Ca’ channels in bovine adrenal glomerulosa cells (29, 30). One of these is similar to the T-type channels described in excitable cells, but is unusually sensitive to dihydropyridine antagonists, and has been suggested to be involved in K+- and AH-stimulated Ca2+ entry. The other population of Ca2+ channels more closely resembles the L-type channels that are present in excitable and many nonexcitable tissues. Several studies have clearly demonstrated that Ca2+influx through VSCC is responsible for the K’-induced rises in [Ca’+]i and aldosterone production, both of which are completely inhibited by dihydropyridine antagonists (2, 14, 15). Furthermore, the dihydropyridine agonist BK 8644 significantly potentiates K’-induced effects in rat adrenal glomerulosa cells, although this action is much less prominent in bovine adrenal glomerulosa cells (12, 13). In contrast to the
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CALCIUM
412
INFLUX
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80
47 + P4 s
20 0
al z
Lz
u
C 10 30 60 120 300 Addition of DuP 753 (set)
c
10 30 60 120 300 Addition of Peptide Antagonists (set)
FIG. 7. Inhibitory effects of AI1 antagonists on the sustained phase of the agonist-induced [Ca’+], response. Upper panel, Time course of the effect of 50 pM DuP 753 on the sustained phase of the [Ca”], elevation in glomerulosa cells stimulated with 2 nM AK The antagonist was added after AI1 at the times indicated. Data are expressed as mean + SEM of at least three measurements at each time point in five experiments. Lower panel, Pooled results obtained by averaging, at the time points indicated, the effects of 1 pM [Sar’,Thr’]AII, 1 pM [Sar’,Ala’] AII, and 1 j.tM [Sar’,Ile’]AII, on plateau-phase [Ca”li responses in cells stimulated with 2 nM AH. Data are the mean + SEM of at least three determinations for each time point and were obtained from four different experiments. In both panels, the left bar (C) shows the mean plateau [Ca”], response to 2 nM AI1 alone.
accepted
role of VSCCs in K’-stimulated glomerulosa cells, such channels participate in the sustained phase of AII-stimulated Ca2+ entry is still in question. In the present work, exposure of bovine glomerulosa cells to nifedipine and verapamil caused a partial but consistent inhibition of the plateau phase of the [Ca’+], response to AII, and a concomitant reduction of the AII-stimulated aldosterone output. These findings, together with the observation that BK 8644 did not potentiate the AU-induced Ca2+ signal, indicate that VSCC make only a modest contribution to Ca2+ movement across the plasma membrane in AII-activated bovine glomerulosa cells. However, the recognition that AI1 stimulates Ca2+ entry mainly through an independent mechanism(s) does not exclude the partial involvement of such channels in agonist-induced Ca 2+ influx and steroid production. In the present study, the inorganic channel blocker, LaC13, inhibited the AII-induced plateau phase of the Ca2+ signal by more than 50%, and also caused a 70% reduction of the AII-induced aldosterone response. These results are in accordance with previous demonstrations that the Ca2+ blocker can abolish the steroidogenic action of AI1 (1, 16), and with
the extent to which
GLOMERULOSA
CELLS
Endo. 1992 Voll31 *No 1
the correlation between cytosolic Ca2+ concentration and steroid production. The greater inhibitory action of lanthanum than nifedipine also indicates that the process which mediates the non-VSCC component of Ca2’ entry is sensitive to inorganic Ca2+ antagonists. Several recent reports have demonstrated a regulatory action of GTP-binding proteins on Ca2+ channel function in various cell types. Studies on guinea pig cardiac myocytes (1 B), and on cultured dorsal root ganglion neurones (19-21), have shown both direct effects of G-proteins on Ca2+ channels and indirect actions through alterations in second messenger formation. In adrenal glomerulosa cells, the AI1 receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein (G,) that mediates AII-induced inhibition of CAMP production, as well as to a PTX-insensitive G-protein (G,) that mediates A&stimulated phospholipid production and [Ca’+], responses (27, 31, 32). A PTXsensitive G-protein has been implicated in the coupling between AI1 receptors and Ca2+ channels in bovine adrenal glomerulosa cells and murine Yl adrenocortical cells (22,33). However, we observed no impairment of AII-stimulated Ca2’ entry in cells treated with a dose of PTX that completely ADP-ribosylated and inactivated the resident Gi-proteins. These findings do not support the proposal that a PTXsensitive G-protein mediates AII-stimulated Ca2+ entry in bovine adrenal cells. The extent to which PTX-insensitive Gproteins participate in calcium channel activation has yet to be determined. Although several reports have contributed to the understanding of receptor-mediated Ca2+ entry through mechanisms involving second messengers or G-proteins, much less is known about Ca2+ channels that are directly activated by agonist-receptor binding (receptor-operated Ca2+ channels) (23). In the course of evaluating the types of Ca2+ channels involved in the AII-evoked sustained phase of Ca2+ response, we investigated the effects of several AI1 antagonists on the calcium entry phase in AII-stimulated glomerulosa cells. Recent studies with selective peptide and nonpeptide antagonists have demonstrated the presence of two subtypes of AI1 receptors (AT1 and AT2) in rat adrenals (34,35). However, only the AT, (Dup 753-sensitive) subtype was detected in adult bovine adrenal glomerulosa cells (36). In our experiments,
the
AT,
selective
nonpeptide
antagonist
(Dup
753),
as well as three peptide antagonists, were added to cells together with AI1 or at short intervals after stimulation by the octapeptide. As expected, the AII-induced Ca2+ signal was abolished by the concomitant addition of AI1 and its antagonists. However, application of antagonists to cells exposed to AI1 for only 10 set resulted in a Ca2+profile in which the initial [Ca”], spike was largely maintained, and the plateau phase was no longer evident. These observations are consistent with the rapidity with which AI1 activates phosphoinositide hydrolysis (9), since addition of antagonists within 10 set only partially reduced intracellular Ca2+ mobilization, but markedly inhibited the subsequent Ca2+ influx. A more detailed analysis of the effects of the antagonists on AII-induced Ca2+ entry was performed at selected time intervals ranging from simulta-
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CALCIUM
INFLUX
IN AII-STIMULATED
neous administration with the agonist to a IO-min delay in their addition. This experiment revealed a progressive loss of ability of the antagonists to inhibit agonist-stimulated Ca2+ entry over a period of 5 min. Thereafter, the AII-evoked Ca*+ entry mechanism was no longer affected by subsequent additions of antagonists for up to 20 min. These findings suggest a close correlation between agonist-receptor sequestration and the sustained phase of the AR-induced [Ca2+li response. In a previous report, [Sar’,Ala8]AII was found to have progressively less inhibitory effect on aldosterone production by bovine glomerulosa cells when added at increasing intervals after AH, suggesting that an activation step in steroidogenesis persists after the termination of receptor occupancy by AI1 (37). Our data indicate that the maintenance of calcium influx is one such step; this could result from the “memory” effect of activated protein kinase C (6, 37), or from prolonged signaling by sequestered or internalized AII-receptor complexes. The binding of hormonal ligands to their receptors is often followed by internalization and degradation of the agonist-receptor complex (38, 39). Although plateau-phase [Ca’+], elevations occur in many cell types in which hormone-receptor internalization has not been implicated in the response to agonist stimulation, at least two reports have demonstrated an association between AII-receptor endocytosis and sustained signal generation (40, 41). In addition, the diacylglycerol response of bovine adrenal glomerulosa cells to AI1 was recently found to persist for up to 75 min after the removal of the agonist, and after the addition of peptide antagonists (42). The results of our study could be explained by the removal of the agonist-receptor complex from the plasma membrane by sequestration and/or endocytosis, a process that appears to be maximal within 5 min in adrenal glomerulosa cells (43). The close correlation between the times required for internalization of the AR-receptor complex and for the development of resistance of Ca*+ influx to blockade by AI1 antagonists suggests that the two phenomena are causally related. Our data indicate that about 5 min are required for the agonist to establish receptor-mediated Ca*+ entry that is no longer influenced by addition of AI1 antagonists. The mechanism(s) involved in this process are not yet clear, and only speculative models can be invoked at this time. These include interaction of the ligand-receptor complex with intracellular components mediating Ca2+ entry through undefined channels, or possibly via the endocytic vesicles themselves, and the continued generation of second messengers that increase membrane permeability to Ca*‘. We have previously observed that the maintenance of AR-induced inositol 1,4,5trisphosphate and [Ca2+li responses in glomerulosa cells appears to be associated with endocytosis of the AII-receptor complex (41), and such a process would not be accessible to blockade by AI1 antagonists. It is also possible that the continued generation of second messenger(s) is independent of the prolonged occupancy of the AI1 receptor. Further studies are needed to evaluate such proposals, and to clarify the relationship between the internalization of ligand-receptor complexes and the maintenance of Ca*+ influx in AIIstimulated glomerulosa cells.
GLOMERULOSA
CELLS
413
Acknowledgments We are grateful to Dr. L. M. Mertz for performance sylation studies in bovine glomerulosa cells.
of ADP-ribo-
References 1. Fakunding 2.
3.
4.
5.
6.
JL, Catt KJ 1980 Dependence
of aldosterone stimulation in adrenal glomerulosa cells on calcium uptake: effects of lanthanum and verapamil. Endocrinology 107:1345-1353 Capponi AM, Lew I’D, Jornot L, Vallotton MB 1984 Correlation between cytosolic free Ca2+ and aldosterone production in bovine adrenal glomerulosa cells, Evidence for a difference in the mode of action of angiotensin II and potassium. J Biol Chem 259:8863-8869 Kojima I, Kojima K, Rasmussen H 1985 Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. J Biol Chem 260:9171-9176 Kojima I, Kojima K, Rasmussen H 1985 Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells. J Biol Chem 261:9177-9184 Capponi AM, Rossier MF, Davies E, Vallotton MB 1988 Calcium stimulates steroidogenesis in permeabilized bovine adrenal cortical cells. J Biol Chem 263:16113-16117
Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H
1989 Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev 10:496-518 7. Kramer RE 1988 Angiotensin II causes sustained elevations in cytosolic calcium in glomerulosa cells. Am J Physiol 255:E338-346 8 Kojima I, Kojima K, Kreutter D, Rasmussen H 1984 The temporal integration of the aldosterone secretory response to angiotensin occurs via two intracellular pathways. J Biol Chem 259:14448-14457 9 BaIla T, Hausdorff WI’, Baukal AJ, Catt KJ 1989 Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin 11. Arch Biochem Biophys 270:398-403
10 Spat A, Balla I, BalIa T, Cragoe Jr EJ, Hajnoczky Gy, Hunyady L
11.
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13.
14.
15.
16.
1989 Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells. J Endocrinol 122:361370 Kojima I, Ogata E 1989 Na-Ca exchanger as a calcium influx pathway in adrenal glomerulosa cells. Biochem Biophys Res Commun 158:1005-1012 Hausdorff WI’, Aguilera G, Catt KJ 1986 Selective enhancement of angiotensin 11- and potassium-stimulated aldosterone secretion by the calcium channel agonist BAY K 8644. Endocrinology 118:869-874 Hausdorff WI’, Catt KJ 1988 Activation of dihydropyridine-sensitive calcium channels and biphasic cytosolic calcium responses by angiotensin II in rat adrenal glomerulosa cells. Endocrinology 123:2818-2826 Aguilera G, Catt KJ 1986 Participation of voltage-dependent calcium channels in the regulation of adrenal glomerulosa function by angiotensin II and potassium. Endocrinology 118:112-l 18 Kojima K, Kojima I, Rasmussen H 1984 Dihydropyridine calcium agonist and antagonist effects on aldosterone secretion. Am J Physiol 247:E645-649
Schiebinger
RJ, Braley LM, Menachery
A, Williams
GH 1986
Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat. J Endocrinol 110:315-325 17. Johnson GL, Dhanasekaran N 1989 The G-protein family and their interaction with receptors. Endocr Rev 10:317-331 18. Yatani A, Codina J, Imoto Y, Reeves JP, Birnbaumer L, Brown AM 1987 A G-protein directly regulates mammalian cardiac calcium channels. Science 238:1288-1292 19. Scott RH, Dolphin AC 1987 Activation of a G protein promotes agonist responses to calcium channel ligands. Nature 330:760-762 20. Ewald DA, Pang I-H, Sternweis PC, Miller RJ 1989 Differential G protein-mediated coupling of neurotransmitter receptors to Cazc channels in rat dorsal root ganglion neurons in vitro. Neuron 2:1185-1193
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414
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21. Dunlap K, Holz GG, Rane SG 1987 G proteins as regulators of ion channel function. TINS 10:241-244 22. Kojima I, Shibata H, Ogata E 1986 Pertussis toxin blocks angiotensin II-induced calcium influx but not inositol triphosphate production in adrenal glomerulosa cells. FEBS Lett 204:347-351 23. Rink TJ, Sage SO 1990 Calcium signaling in human platelets. Annu Rev Physiol 52:431-449 24. Balla T, Baukal AJ, Guillemette G, Catt KJ 1988 Multiple pathways of inositol polyphosphate metabolism in angiotensin-stimulated adrenal glomerulosa cells. J Biol Chem 263:4083-4091 25. Ely JA, Ambroz C, Baukal AJ, Christensen SB, Balla T, Catt KJ 1991 Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. J Biol Chem 266:1863518641 26. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450 27. Hausdorff WP, Sekura RD, Aguilera G, Catt KJ 1987 Control of aldosterone production by angiotensin II is mediated by two guanine nucleotide regulatory proteins. Endocrinology 120:1668-1678 28. Balla T, Varnai l’, Ho110 Z, Spat A 1990 Effects of high potassium concentration and dihydropyridine Ca’+-channel agonist on cytoplasmic Ca’+ and aldosterone production in rat adrenal glomerulosa cells. Endocrinology 127:815-822 29. Matsunaga H, Yamashita N, Maruyama Y, Kojima I, Kurokawa K 1987 Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells. Biochem Biophys Res Commun 149:1049-1054 30. Cohen CJ, McCarthy RT, Barrett PQ, Rasmussen H 1988 Ca channels in adrenal glomerulosa cells: K’ and angiotensin II increase T-type Ca channel current. Proc Nat1 Acad Sci USA 85:2412-2416 31. Catt KJ, BaIla T, Baukal AJ, Hausdorff WP, Aguilera G 1988 Control of glomerulosa cells function by angiotensin II: transduction by G-proteins and inositol polyphosphates. Clin Exp Pharmcol Physiol 15:501-515 32. Baukal AJ, Balla T, Hunyady L, Hausdorff W, Guillemette G, Catt KJ 1988 Angiotensin II and guanine nucleotides stimulate formation of inositol 1,4,5-trisphosphate and its metabolites in permeabilized adrenal glomerulosa cells. J Biol Chem 263:6087-
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CELLS
Endo. Vol131.
1992 No 1
6092 33. Hescheler J, Rosenthal W, Hinsch KD, Wulfern M, Trautwein W, Schultz G 1988 Angiotensin II-induced stimulation of voltagedependent Ca*+ currents in an adrenal cortical cell line. EMBO J 7~619-624 34. Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wang PC, Wexler RR, Johnson AL, Timmermans EBMWM 1989 Identification of angiotensin II receptor subtypes. Biochem Biouhvs Res Commun 165:196-203 35. Chang RSL: Lotti VJ 1989 Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands. Mol Pharmacol 29:347-351 36. Balla T, Baukal AJ, Eng S, Catt KJ 1991 Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla. Mol Pharmacol 40:401-406 37. Capponi AM, Lew PD, Vallotton MB 1987 Quantitative analysis of the cytosolic-free-Ca’+-dependency of aldosterone production in bovine adrenal glomerulosa cells. Biochem J 247:335-340 38. Catt KJ, Harwood JP, Aguilera G, Dufau ML 1979 Hormonal regulation of peptide receptors and target cell responses. Nature 280:109-116 39. Crozat A, Penhoat A, Saez JM 1986 Processing of angiotensin II (A-II) and (Sari, Ala8)A-II by cultured bovine adrenalcortical cells, Endocrinology 118:2312-2318 40. Griendling KK, Delafontaine P, Rittenhause SE, Gimbrone Jr MA, Alexander RW 1987 Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells. J Biol Chem 262:1455514562 41. Hunyady L, Merelli F, Baukal AJ, Balla T, Catt KJ 1991 Agonistinduced endocytosis and signal generation in adrenal glomerulosa cells. J Biol Chem 266:2783-2788 42. Bollag WB, Barrett PQ, Isales CM, Rasmussen H 1991 Angiotensin II-induced changes in diacylglycerol levels and their potential role in modulating the steroidogenic response. Endocrinology 128:231241 43. Bianchi C, Gutkowska J, De Lean A, Ballak M, Anand-Srivastava MB, Genest J, Cantin M 1986 Fate of [‘251]angiotensin II in adrenal zona glomerulosa cells. Endocrinology 118:2605-2607
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Vol. 131, No. 1 Printed m Ii S A
Society
Angiotensin II Receptor-Mediated Calcium Bovine Adrenal Glomerulosa Cells* CLARA
AMBROZ
Endocrinology Development,
AND
KEVIN
and Reproduction National Institutes
Influx
in
J. CATT Research Branch, National Institute of Child Health of Health, Bethesda, Maryland 20892
ABSTRACT The cytoplasmic calcium ([Ca”],) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca’+ mobilization, followed by a sustained plateau phase that is dependent on Ca” entry from the extracellular fluid. In Furaloaded cells, the calcium channel antagonists, nifedipine (1 pM) and verapamil (20 PM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca”], when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca’+],. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca’+ channel blocker, LaCl,, (10 @M), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca” signal was not affected by treatment with pertussis
and Human
toxin (100-300 rig/ml for 12 h). The prior addition of specific AIIantagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sarl,ThrR]AII, [Sar’,Ala”]AII, and [Sar’,Ile”]AII) completely inhibited the AII-induced Ca’+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 set to 5 min after AI1 caused progressively less attenuation of the sustained Ca”+ signal. After 5 min, addition of antagonists did not inhibit the agonistinduced Ca’+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca’+], could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca’+ influx despite receutor blockade. It is nossible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca’+], response. (Endocrinology 131: 408414,1992)
A
NGIOTENSIN II (AII), a major physiological regulator of aldosterone production by adrenal glomerulosa cells, stimulates steroidogenesis and hormone secretion by activation of phosphoinositide hydrolysis and elevation of cytoplasmic Ca2+ concentration ([Ca’+],) (l-7). Although the role of Ca2’ as an intracellular messenger for the action of AI1 is well established, the mechanisms responsible for the agonist-induced increase in [Ca’+], are not yet clear. The secretory response to agonist stimulation in adrenal glomerulosa cells is accompanied by an initial transient rise in [Ca’+], that is largely derived from mobilization of intracellular Ca2+ stores, and is followed by a prolonged plateau phase that depends on Ca2+ entry across the plasma membrane. While the initial transient increase in [Ca’+], is coincident with and attributable to the early peak of Ins(1,4,5)P, production (6, 8, 9), the entry pathway(s) that provide Ca2+ to sustain the plateau phase of the AII-induced [Ca’+], response are not well defined (4, 6, 7, 10, 11). There are species differences in the extent to which voltage-sensitive calcium channels (VSCC) participate in AIIinduced [Ca2+li responses and aldosterone production in rat and bovine glomerulosa cells. These are exemplified by the
ability of the dihydropyridine calcium channel agonist, BAY K 8644 (BK 8644), to enhance cytoplasmic Ca2+ and aldosterone secretion in AII-stimulated rat but not in bovine glomerulosa cells (12, 13). Several reports have shown that AIIinduced aldosterone production is reduced by antagonists of VSCC in both species (1, 2, 14-16), but the enhanced Ca2+ entry observed in AII-stimulated bovine glomerulosa cells is less affected by such compounds (2, 13). Recent evidence has indicated that guanine nucleotide regulatory (G) proteins are involved in the regulation of Ca2+ channel function in several cell types (17-21), and it has been suggested that AII-mediated Ca2+ influx is dependent on the action of a regulatory G-protein (22). In addition, receptor-operated Ca2+ channels have been proposed to mediate Ca2+ entry through pathways other than L-type Ca2+ channels; however, there is relatively little evidence to support the existence of such channels (23). In the present study, we examined the potential mechanisms of AII-mediated Ca2+ entry in bovine glomerulosa cells to better define their participation in the maintenance of agonist-stimulated [Ca’+] signaling responses. Materials
Received December 19, 1991. Address correspondence and requests for reprints to: Dr. Kevin J. Catt, Room Bl-L400, Building 10, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marvland 20892. *This investigation was supported by The National Institute of Child Health and Human Develoument, National Research Service Award HD-07383-03, from the EAdocrinology and Reproduction Research Branch.
and Methods
Materials Cell culture media were prepared by the NIH Media Supply Unit (Bethesda, MD). AII, [Sar’,Th;‘]AII, [Sar’;IleR]AII, and [Sar’,Ala’jAiI were purchased from Peninsula Laboratories, Inc. (San Carlos, CA). The nonpeptide antagonist, DuP 753, was kindly provided by Dr.‘P. C. Wong (DuPont, Wilmington, DE). [a?‘]NAD (5 mCi/ml) was obtained from DuPont-New England Nuclear (Boston, MA). lZ51-AII was obtained from Hazleton Labs (Vienna, VA), and 125-I aldosterone RIA kits from
408
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CALCIUM
INFLUX
IN AII-STIMULATED
Diagnostic Product Corp. (Los Angeles, CA). Fura-2/AM was obtained from Molecular Probes, Inc. (Eugene, OR). All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO).
Preparation
of bovine glomerulosa
cells
Bovine adrenal glomerulosa cells were prepared and cultured as described (24) with the exception that metyrapone (5 PM) was included in the culture medium. Cells were plated in 6-well culture dishes at a density of 1 million cells per ml for cytoplasmic Ca’+ measurements and in 24-well plates at a density of 0.5-l million per well for steroid production assays, and were maintained in culture for 2 or 3 days. One day before Furaloading or aldosterone production experiments, the culture medium was changed to a serum-free solution. For cytoplasmic Ca*+ assays, the cells were collected and incubated with Fura-2/AM (final concentration: 1 PM) as described previously (25).
Measurement
of cytoplasmic
calcium
After incubation with Fura-2/AM the cells were washed twice in a modified Krebs-Ringer solution (KRB) (1.8 rnM Ca&, 2.4 mM KCl, 123 rnM NaCl, 0.8 mM MgC12, 1.2 mM KH2POI, 5 mM NaHC03, and 10 mM glucose) supplemented with 20 mM sodium HEPES (pH 7.4), resuspended in the same buffer at a concentration of 2-4 million cells per 100 ~1 and kept at room temperature until analyzed for [Ca”+], responses. For each [Ca’+], determination a loo-p1 aliquot of washed Fura-2-loaded cells was transferred to a quartz cuvette containing 1.9 ml modified KRB. Analysis of [Ca”+li responses in the absence of extracellular Ca*+ was performed by resuspension of the cells in a cuvette containing KRB without CaClz immediately before spectrofluorimetric analysis. All fluorescence determinations were performed in a PTI-Deltascan spectrofluorometer (Photon Technology International Inc., South Brunswick, NJ) connected to a microcomputer for data storage and analysis. The cuvette holder was equipped with a magnetic stirrer and maintained at a constant temperature of 30 C. Data were collected using dual wavelength excitation at 340 and 380 nm and an emission wavelength of 500 nm (slit width was 4 nm for excitation and emission). Cytoplasmic Ca” concentrations were calculated from the fluorescence ratio according to the method of Grynkiewicz et al. (26) and the values were corrected for cell autofluorescence using software provided with the PTI-Deltascan system.
Aldosterone
production
Before aldosterone production measurements, cultured bovine adrenal glomerulosa cells were washed twice and incubated for 1 h in bicarbonate-buffered modified M-199 at 37 C in an atmosphere of 5% C02/95% O2 to remove metyrapone. At the end of this period the medium was replaced and the cells were incubated with selected concentrations of reagents for 15 min to 2 h at 37 C. Cells were then placed on ice, and the medium was collected and stored at -20 C for aldosterone analysis by RIA.
GLOMERULOSA
Role of dihydropyridine-sensitive induced [Ca2+], responses
ADP-Ribosylation studies were performed according to a previously described protocol (27). Briefly, glomerulosa membranes (20-40 pg) from control cells and pertussis toxin (PT)-treated cells were added to tube containing 0.1 ml 100 rnM Tris (pH 8.0), 10 rnM thymidine, 1 mM ATP, 100 KM GTI’, 2.5 mM MgCl,, 1 mM EDTA, 10 mM dithiothreitol, 2 FCi [a-?‘]NAD, and l-2 fig activated PT. Samples were incubated for 3060 min at 37 C, and the reaction was terminated by addition of 200 ~1 gel sample buffer containing 4% (wt/vol) sodium dodecyl sulfate (SDS), followed by boiling for 5 min. Proteins were separated by SDS-polyacrylamide gel (12%) electrophoresis, followed by autoradiography.
Statistical
analysis
Student’s t test was used to determine between means.
the significance
of differences
calcium channels
in AII-
As illustrated in Fig. 1, the rise in [Ca’+]i induced by 100 nM AI1 was biphasic, with a transient peak that was only slightly reduced in the absence of extracellular Ca*+, followed by a sustained phase of elevated [Ca’+], that was completely abolished in Ca2+-free medium. The addition of 20 PM verapamil to cells before stimulation by 2 nM AII, or during the sustained phase of the AII-evoked Ca2+ response, had no consistent effect on the peak height but caused a partial decrease of the plateau phase (Fig. 2A). Preincubation of the cells with 1 PM nifedipine, or addition of the VSCC antagonist during the sustained phase of the AII-induced [Ca*+]i response, had the same partial inhibitory effect as verapamil (Fig. 2B). The addition of 50-100 nM BK 8644 to bovine adrenal glomerulosa cells caused a slow and minor increase in basal [Ca’+J, but did not enhance the subsequent Ca*+ response evoked by 2 nM AII. Addition of the dihydropyridine agonist during the plateau phase caused a small and additive increase in the Ca*+ signal (Fig. 2C). These findings, which were confirmed in five different experiments, were consistent with previous observations (2, 12) that the majority of the rise in cytoplasmic Ca 2+ induced by AI1 in bovine adrenal glomerulosa cells is independent of VSCC activation. In 2-h incubations, nifedipine (1 FM) inhibited the aldosterone production induced by AI1 (1 nM) by 30%, from 1186 + 156 to 832 + 85 pg/ml (means f SD, n = 4, P < 0.05) (data not shown). Effects of inorganic
calcium channel
blockade
The ability of the inorganic calcium channel blocker, LaC&, to reduce or abolish the agonist-induced Ca*+ entry process was evaluated in AH-stimulated glomerulosa cells. The results obtained from 10 determinations performed in 3 different cell preparations are summarized in Fig. 3. Addition of 600 r
C
studies
409
Results
5 400 ADP-Ribosylation
CELLS
,-GN+ ci 200
Cont & Ca2 ’ -free
-D
I 0
All 1100 nM1
100
200 Time
300
(set)
Fro. 1. Extracellular Ca’+ dependence of the AII-induced sustained [Ca”+], response. Fura 2-loaded cells were centrifuged and resuspended in Ca”+-free medium immediately before the beginning of the recording (time 0), and 100 nM AI1 was added to the cuvette within 2 min. The data are typical traces from experiments repeated more than six times.
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CALCIUM
t
I
0 -
INFLUX
I
I
I
IN AII-STIMULATED
L
0
,I
I
I
200 Time (set)
‘Lo
I
I
300
400
FIG. 3. Inhibition of the sustained phase of the AII-stimulated [Ca”], response by LaC13. The inorganic Ca2+ channel blocker (10 PM) was added before (truce a) or during the plateau phase (truce b) of [Ca”li responses stimulated by 2 nM AII. The traces shown are representative of 10 determinations made in 3 different experiments.
Nif.
I
1992 No 1
La3+ I
0’
1
Endo. Vol131.
All
La3 ’
mrB
‘(%-G-i
CELLS
I
100200300400500600 Time (set)
I
GLOMERULOSA
1
100200300400500600 Time (set)
400 5
r Normal
Medium
300
c F 200 N m o
100 t
I
0 Time
(set)
FIG. 2. Effect of organic Ca2+ channel antagonist and agonist compounds on AII-stimulated cells. A, Verapamil (20 FM) was added at time 0 (truce a) or during the sustained phase of the Ca*+ response to 2 nM AI1 (trace b); B, 1 PM nifedipine was added before (trace a) or after (truce b) the addition of 2 nM AI1 at the times indicated. This concentration of nifedipine caused a slight depression of the baseline in one of four cell preparations studied. C, BK 8644 (BK) (100 nM) was added when indicated by the arrow to cells subsequently stimulated by 2 nM AI1 (truce a) or to cells previously stimulated with 2 nM AI1 (truce 6). Similar results were obtained with a lower dose of BK 8644 (50 nM). The traces shown in the panels represent the results of three (A), four (B) or five (C) different experiments.
10 PM LaC13, either before or after stimulation of the cells by 2 nM AII, did not affect the initial peak response but reduced the sustained phase of the [Ca’+], response by up to 50%. Higher concentrations of LaC4 (up to 20 PM) abolished up to 80% of the AII-induced plateau phase of the calcium response. However, such doses also caused depression of the basal [Ca’+]i level in unstimulated cells (data not shown). In 2-h incubations, LaCL (10 PM) caused a significant (P C 0.01) reduction in AII-stimulated aldosterone production, which decreased from 166 + 23 pg/ml in control cells to 55 & 20 pg/ml (mean f SD, n = 3, P < 0.01). Role of G-proteins
in AII-induced
calcium entry
To evaluate the role of Gi or G, proteins in the mechanism of agonist-stimulated calcium entry in bovine adrenal glomerulosa cells, we examined the effect of pertussis toxin (PTX) on the calcium response.Cells pretreated for 12 h with
400
I
100 Time
Ca*+
- Free
I
200 (set)
300
I
I
Medium
r
I
I
200 300 100 Time (set) FIG. 4. Cytoplasmic calcium responses to AI1 in PTX-treated glomerulosa cells. Upperpanel, Representative tracings of the [Ca”+], response to 2 nM AI1 in cells pretreated with PTX (300 mg/ml for 12 h us. control cells. Lower doses of PTX (100 rig/ml for 12 h) had similar effects. Lower panel, Experimental conditions were the same as in the upper panel with the exception that the cells were centrifuged and resuspended in Ca*+-free medium at the beginning of the recording. These results (upper and lower panel) summarize observations from six different experiments. 0
100-300 rig/ml PTX showed no change in the Ca2+profile evoked by 2 nM AI1 (Fig. 4, top). We sometimesobserved a slight reduction of the [Ca’+], peak which appeared to be dependent on the presence of extracellular Ca2+ (Fig. 4, bottom). However, this effect was present only in two of the six experiments performed with PTX. Likewise, in accordance with previous reports (27), no inhibitory effect on aldosterone production was observed (data not shown). The effectiveness of the PTX treatment was confirmed by SDS-polyacrylamide
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CALCIUM
INFLUX
IN AII-STIMULATED
GLOMERULOSA
mr
gel electrophoresis of solubilized membrane proteins prepared from cells preincubated with the lowest PTX concentration used (100 @ml). Autoradiograms of such gels revealed that ADP-ribosylation of a 41-kilodalton protein corresponding to the a-subunit of Gi and G, was reduced by about 95% in membranes from PTX-treated cells vs. control cells (Fig. 5).
DuP 753 (50 FM) Added
t
Effects of receptor antagonists
on All-stimulated
calcium entry
Evidence for the participation of agonist-activated Ca2+ channels in the maintenance of AII-induced increases in [Ca’+], was sought by exposing AII-stimulated glomerulosa cellsto specific AI1 antagonists at selectedtimes after addition of the agonist. As shown in Fig. 6, both DuP 753 and [Sar’, Ile*]AII caused marked inhibition of the plateau phase of the Ca2+ signal when added soon after 2 nM AII. The initial [Ca’+]i spike was maintained even when the time interval between the administration of the agonist and antagonist was only 10 set, but the subsequent plateau phase was abolished or attenuated. Addition of the antagonists at later time points caused progressively less inhibition of the sustained phase of Ca2+entry, and had no effect on the [Ca’+], plateau when performed more than 5 min after stimulation with AII. The concomitant administration of agonist and antagonist, as expected, caused complete inhibition of the AII-stimulated Ca2+signal (not shown). The data shown in Fig. 7 represent the plateau [Ca’+], levels reached after addition of DuP 753 and three peptide antagonists, all of which gave similar results. They are expressed as the mean + SEM of at least three different measurementsat each time point, and demonstrate the progressive lossof inhibition of the AIIinduced [Ca2’J response by delayed addition of the AI1 antagonists, even in concentrations up to 25,000-fold molar excess. Discussion It is well established that AI1 and extracellular potassium (K+), two primary regulators of aldosterone production by adrenal zona glomerulosa cells, exert their stimulatory effects through an increase in cytoplasmic Ca2+ concentration (6, recent review). Although the Ca2+ entry through which K+ evokes aldosterone production is clearly dependent on acti-
123456
36-, FIG. 5. ADP-Ribosylation of membrane fractions from control and PTX-treated bovine adrenal glomerulosa cells. Pretreatment of 3-day glomerulosa cell cultures with PTX (100 rig/ml for 12 h) inhibited subsequent in vitro ADP-ribosylation of a M, = 41,000 protein. Membrane fractions were prepared from control cultures (lanes 1, 3, 5) and PTX-treated cultures (lanes 2, 4, 6); the same cell preparations were used for the analysis of AII-induced [Caz+li responses.
411
CELLS
I
0
300
=
r I I
[Sar’,
lle*lA11
AI1 (2 nM) +
I
loo
I
200 300 Time (set)
at: (set)
I
400
(I FM)
I
I
I
I
200 300 400 Time (sec) FIG. 6. Inhibitory effects of AI1 antagonists on agonist-induced lCa*‘l, responses. Upper panel, Progressive loss of the inhibitory effect of Dup 753 (50 pM) added at increasing intervals after 2 nM AII; lower wanel. similar loss of inhibition bv 1 uM lSar’.Ile”lAII added at increas: mg intervals after 2 nM AD. The times of addition of antagonist are indicated by the arrows, and at the side of each record. The control traces were identical with the 300-set traces and are not shown. The data are representative of results obtained in nine experiments. 0
loo
vation of VSCC, the mechanism(s)responsible for AII-stimulated Ca2+movement acrossthe cell membrane are not yet understood (2, 3, 7, 10, 13, 14, 28). There is ample evidence that the rise in [Ca2+]i which follows activation of the AI1 receptor is caused by at least two distinct processes:a transient release of Ca2+ into the cytoplasm from intracellular stores, and an accompanying sustained increase of Ca2+ influx across the cell membrane. The rapid and transient [Ca2+]iresponseis known to be mediated by phosphoinositide hydrolysis and increased production of Ins(1,4,5)P3 (6), but relatively little is known about the Ca2+ entry process. Recent studies using patch clamp techniques have indicated the presence of two distinct types of Ca’ channels in bovine adrenal glomerulosa cells (29, 30). One of these is similar to the T-type channels described in excitable cells, but is unusually sensitive to dihydropyridine antagonists, and has been suggested to be involved in K+- and AH-stimulated Ca2+ entry. The other population of Ca2+ channels more closely resembles the L-type channels that are present in excitable and many nonexcitable tissues. Several studies have clearly demonstrated that Ca2+influx through VSCC is responsible for the K’-induced rises in [Ca’+]i and aldosterone production, both of which are completely inhibited by dihydropyridine antagonists (2, 14, 15). Furthermore, the dihydropyridine agonist BK 8644 significantly potentiates K’-induced effects in rat adrenal glomerulosa cells, although this action is much less prominent in bovine adrenal glomerulosa cells (12, 13). In contrast to the
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CALCIUM
412
INFLUX
IN AII-STIMULATED
80
47 + P4 s
20 0
al z
Lz
u
C 10 30 60 120 300 Addition of DuP 753 (set)
c
10 30 60 120 300 Addition of Peptide Antagonists (set)
FIG. 7. Inhibitory effects of AI1 antagonists on the sustained phase of the agonist-induced [Ca’+], response. Upper panel, Time course of the effect of 50 pM DuP 753 on the sustained phase of the [Ca”], elevation in glomerulosa cells stimulated with 2 nM AK The antagonist was added after AI1 at the times indicated. Data are expressed as mean + SEM of at least three measurements at each time point in five experiments. Lower panel, Pooled results obtained by averaging, at the time points indicated, the effects of 1 pM [Sar’,Thr’]AII, 1 pM [Sar’,Ala’] AII, and 1 j.tM [Sar’,Ile’]AII, on plateau-phase [Ca”li responses in cells stimulated with 2 nM AH. Data are the mean + SEM of at least three determinations for each time point and were obtained from four different experiments. In both panels, the left bar (C) shows the mean plateau [Ca”], response to 2 nM AI1 alone.
accepted
role of VSCCs in K’-stimulated glomerulosa cells, such channels participate in the sustained phase of AII-stimulated Ca2+ entry is still in question. In the present work, exposure of bovine glomerulosa cells to nifedipine and verapamil caused a partial but consistent inhibition of the plateau phase of the [Ca’+], response to AII, and a concomitant reduction of the AII-stimulated aldosterone output. These findings, together with the observation that BK 8644 did not potentiate the AU-induced Ca2+ signal, indicate that VSCC make only a modest contribution to Ca2+ movement across the plasma membrane in AII-activated bovine glomerulosa cells. However, the recognition that AI1 stimulates Ca2+ entry mainly through an independent mechanism(s) does not exclude the partial involvement of such channels in agonist-induced Ca 2+ influx and steroid production. In the present study, the inorganic channel blocker, LaC13, inhibited the AII-induced plateau phase of the Ca2+ signal by more than 50%, and also caused a 70% reduction of the AII-induced aldosterone response. These results are in accordance with previous demonstrations that the Ca2+ blocker can abolish the steroidogenic action of AI1 (1, 16), and with
the extent to which
GLOMERULOSA
CELLS
Endo. 1992 Voll31 *No 1
the correlation between cytosolic Ca2+ concentration and steroid production. The greater inhibitory action of lanthanum than nifedipine also indicates that the process which mediates the non-VSCC component of Ca2’ entry is sensitive to inorganic Ca2+ antagonists. Several recent reports have demonstrated a regulatory action of GTP-binding proteins on Ca2+ channel function in various cell types. Studies on guinea pig cardiac myocytes (1 B), and on cultured dorsal root ganglion neurones (19-21), have shown both direct effects of G-proteins on Ca2+ channels and indirect actions through alterations in second messenger formation. In adrenal glomerulosa cells, the AI1 receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein (G,) that mediates AII-induced inhibition of CAMP production, as well as to a PTX-insensitive G-protein (G,) that mediates A&stimulated phospholipid production and [Ca’+], responses (27, 31, 32). A PTXsensitive G-protein has been implicated in the coupling between AI1 receptors and Ca2+ channels in bovine adrenal glomerulosa cells and murine Yl adrenocortical cells (22,33). However, we observed no impairment of AII-stimulated Ca2’ entry in cells treated with a dose of PTX that completely ADP-ribosylated and inactivated the resident Gi-proteins. These findings do not support the proposal that a PTXsensitive G-protein mediates AII-stimulated Ca2+ entry in bovine adrenal cells. The extent to which PTX-insensitive Gproteins participate in calcium channel activation has yet to be determined. Although several reports have contributed to the understanding of receptor-mediated Ca2+ entry through mechanisms involving second messengers or G-proteins, much less is known about Ca2+ channels that are directly activated by agonist-receptor binding (receptor-operated Ca2+ channels) (23). In the course of evaluating the types of Ca2+ channels involved in the AII-evoked sustained phase of Ca2+ response, we investigated the effects of several AI1 antagonists on the calcium entry phase in AII-stimulated glomerulosa cells. Recent studies with selective peptide and nonpeptide antagonists have demonstrated the presence of two subtypes of AI1 receptors (AT1 and AT2) in rat adrenals (34,35). However, only the AT, (Dup 753-sensitive) subtype was detected in adult bovine adrenal glomerulosa cells (36). In our experiments,
the
AT,
selective
nonpeptide
antagonist
(Dup
753),
as well as three peptide antagonists, were added to cells together with AI1 or at short intervals after stimulation by the octapeptide. As expected, the AII-induced Ca2+ signal was abolished by the concomitant addition of AI1 and its antagonists. However, application of antagonists to cells exposed to AI1 for only 10 set resulted in a Ca2+profile in which the initial [Ca”], spike was largely maintained, and the plateau phase was no longer evident. These observations are consistent with the rapidity with which AI1 activates phosphoinositide hydrolysis (9), since addition of antagonists within 10 set only partially reduced intracellular Ca2+ mobilization, but markedly inhibited the subsequent Ca2+ influx. A more detailed analysis of the effects of the antagonists on AII-induced Ca2+ entry was performed at selected time intervals ranging from simulta-
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CALCIUM
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neous administration with the agonist to a IO-min delay in their addition. This experiment revealed a progressive loss of ability of the antagonists to inhibit agonist-stimulated Ca2+ entry over a period of 5 min. Thereafter, the AII-evoked Ca*+ entry mechanism was no longer affected by subsequent additions of antagonists for up to 20 min. These findings suggest a close correlation between agonist-receptor sequestration and the sustained phase of the AR-induced [Ca2+li response. In a previous report, [Sar’,Ala8]AII was found to have progressively less inhibitory effect on aldosterone production by bovine glomerulosa cells when added at increasing intervals after AH, suggesting that an activation step in steroidogenesis persists after the termination of receptor occupancy by AI1 (37). Our data indicate that the maintenance of calcium influx is one such step; this could result from the “memory” effect of activated protein kinase C (6, 37), or from prolonged signaling by sequestered or internalized AII-receptor complexes. The binding of hormonal ligands to their receptors is often followed by internalization and degradation of the agonist-receptor complex (38, 39). Although plateau-phase [Ca’+], elevations occur in many cell types in which hormone-receptor internalization has not been implicated in the response to agonist stimulation, at least two reports have demonstrated an association between AII-receptor endocytosis and sustained signal generation (40, 41). In addition, the diacylglycerol response of bovine adrenal glomerulosa cells to AI1 was recently found to persist for up to 75 min after the removal of the agonist, and after the addition of peptide antagonists (42). The results of our study could be explained by the removal of the agonist-receptor complex from the plasma membrane by sequestration and/or endocytosis, a process that appears to be maximal within 5 min in adrenal glomerulosa cells (43). The close correlation between the times required for internalization of the AR-receptor complex and for the development of resistance of Ca*+ influx to blockade by AI1 antagonists suggests that the two phenomena are causally related. Our data indicate that about 5 min are required for the agonist to establish receptor-mediated Ca*+ entry that is no longer influenced by addition of AI1 antagonists. The mechanism(s) involved in this process are not yet clear, and only speculative models can be invoked at this time. These include interaction of the ligand-receptor complex with intracellular components mediating Ca2+ entry through undefined channels, or possibly via the endocytic vesicles themselves, and the continued generation of second messengers that increase membrane permeability to Ca*‘. We have previously observed that the maintenance of AR-induced inositol 1,4,5trisphosphate and [Ca2+li responses in glomerulosa cells appears to be associated with endocytosis of the AII-receptor complex (41), and such a process would not be accessible to blockade by AI1 antagonists. It is also possible that the continued generation of second messenger(s) is independent of the prolonged occupancy of the AI1 receptor. Further studies are needed to evaluate such proposals, and to clarify the relationship between the internalization of ligand-receptor complexes and the maintenance of Ca*+ influx in AIIstimulated glomerulosa cells.
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Acknowledgments We are grateful to Dr. L. M. Mertz for performance sylation studies in bovine glomerulosa cells.
of ADP-ribo-
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