0013-7227/90/1272-0541$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 2 Printed in U.S.A.
Cytosolic Calcium and Aldosterone Response Patterns of Rat Adrenal Glomerulosa Cells Stimulated by Vasopressin: Comparison with Angiotensin II* STEPHEN J. QUINN, PETER ENYEDIf, DOUGLAS L. TILLOTSON, AND GORDON H. WILLIAMS Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118; and the Endocrine-Hypertension Division, Brigham and Women's Hospital and Harvard Medical School (S.J.Q., P.E., G.M. WJ, Boston, Massachusetts 02115
ABSTRACT. Cytosolic calcium (Cai) responses to arginine vasopressin (AVP) and angiotensin-II (Ang II) were examined in single rat adrenal zona glomerulosa (ZG) cells by monitoring fura-2 fluorescence with microspectrofluorimetry. ZG cells displayed dose-dependent Cai responses to a wide range of AVP and Ang II concentrations, starting from a threshold of 1 nM for AVP and less than 5 pM for Ang II. A dose-dependent delay of the onset of the Ca; response was observed with both hormones. The response delay for Ang II was consistently briefer than that for the same concentration of AVP, showing a 2-3 log unit separation in the dose-response relations. After the delay, cells typically responded with an abrupt increase in Cai( which peaked within 15 sec. The amplitude of the peak Ca; rise showed little dependency on AVP or Ang II concentration. At most AVP concentrations, the response consisted of Ca; oscillations, with apparent fusion of these Caj oscillations at the highest AVP concentrations (1-0.1 pM). Similar oscillatory behavior was found with stimulation by much lower Ang II concentrations (0.5 nM to 5 pM). There appeared to be a 2-3 log unit shift in
the sensitivity toward AVP and Ang II when Ca; responses were compared. Sixty percent of ZG cells were responsive to AVP, while more than 90% displayed an elevation of Caj with Ang II. The Cai and steroid responses to 100 nM AVP and 100 pM Ang II were compared, since these two doses are reported to stimulate the phosphoinositide system to a similar extent. Individual ZG cells tested with both hormones responded with equivalent peak Ca; changes, but a slightly longer response delay for AVP. The mean Cai response and aldosterone production for each secretagogue displayed parallel kinetics during 30-min stimulations. After initial oscillations, the Caj response returned to control values within 15 min of 100 nM AVP application. Likewise, the steroid output was transient. In contrast, 100 pM Ang II produced maintained Ca; oscillations as well as a sustained and substantially greater aldosterone production for the same period of application. In conclusion, the disparate steroidogenic effects of AVP and Ang II appear to result from distinctly different Caj responses elicited during maintained secretagogue stimulation. (Endocrinology 127: 541-548,1990)
T 7ASOPRESSIN (AVP) and angiotensin-II (Ang II) V are aldosterone secretagogues for adrenal zona glomerulosa (ZG) cells. While the steroidogenic action of AVP is substantially less potent than that of Ang II in most studies, these secretagogues appear to act through similar transduction pathways (1-5). The binding of both AVP and Ang II to specific membrane receptors results in hydrolysis of phosphatidylinositol 4,5-bisphosphate and the production of two initial intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins-
1,4,5-P3) (1-6). Ins-1,4,5-P3is known to mobilize calcium from intracellular stores in ZG cells (6, 7), and the resultant increases in cytosolic calcium (Cai) probably serve as an intracellular message for the stimulation of aldosterone production. Ca; responses to Ang II stimulation have been demonstrated in ZG cell populations (8-11) and in single ZG cells (12, 13); however, no Ca; studies of AVP stimulation have been reported. Studies of aldosterone stimulation have revealed differences in the steroidogenic actions of AVP and Ang II. At its maximal dose, AVP typically stimulates substantially less aldosterone (and Ins-1,4,5-P3) production than Ang II (1, 2, 5). Furthermore, the kinetics of the secretory responses are also distinct. In superfusion studies, continuous application of Ang II produces a sustained aldosterone response, while AVP only transiently stimulates steroid output (1, 5). Paradoxically, the transient aldosterone response pattern of AVP is associated with a long-lasting stimulation of the phosphoinositide system
Received February 19, 1990. Address all correspondence and requests for reprints to: Dr. Stephen J. Quinn, Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. * This work was supported in part by the following NIH grants: Specialized Center of Research (SCOR) in Hypertension Grant HL36568, Training Grant HL-07609, HL-36420, HL-42354, and DK40127. The data were analyzed using a CLINFO system supported by Grant 2635 from the Division of Research Resources. t Present address: Department of Physiology, Semmelweis University, Budapest, Hungary.
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Ca; RESPONSES OF ADRENAL CELLS TO AVP AND ANG II
542
(5). While these aldosterone response patterns may result from differences in the pathway of inositol phosphate metabolism, variations in the secretagogue-mediated Ca; signal may also be involved. Thus, the purpose of the present study was to compare the Ca; and steroid responses to AVP and Ang II in superfused rat ZG cells.
Materials and Methods Isolated rat ZG cells were prepared as previously described (14). Briefly, adrenal capsules of female Sprague-Dawley rats were digested with collagenase and DNAse dissolved in the incubation medium described below, followed by mechanical dispersion of the tissue. Aldosterone production of the isolated ZG cells was examined in a system of superfusion columns (15). The incubation solution was HEPES-buffered medium 199 (Gibco, Grand Island, NY) with 3.6 mM KC1, pH 7.4, completed with 2 mg/ml BSA. Cells from six rats were loaded into each column and superfused at a flow rate of 0.2 ml/min. The columns were superfused with control medium for 1 h and then stimulated with AVP or Ang II for 55-60 min. Effluent fractions were collected every 2-5 min and analyzed for aldosterone by a RIA kit (Diagnostic Products, Los Angeles, CA). The transit time of the superfusion system (6 min) was determined by superfusion of radiolabeled Ang II and was taken into consideration when determining the kinetics of aldosterone production. Measurement of Cat Dispersed ZG cells were incubated for 20 min with the acetoxymethyl ester of fura-2 (Molecular Probes, Eugene, OR) at a concentration of 5 ^M in Eagle's Minimal Essential Medium including HEPES (20 mM), CaCl2 (1.25 mM), MgSO4 (0.5 mM), and BSA (0.1%), pH 7.47. The cell suspensions were washed and used 1-4 h after loading with the fura-2 ester. A suspension of adrenal cortical cells was placed in a superfusion chamber, allowed to adhere to its glass coverslip bottom, and continuously superfused with a warmed (35-37 C) solution (NaCl, 120 mM; KC1, 4 mM; Na2PO4, 1.2 mM; CaCl2,1.25 mM; MgSO4, 0.5 mM; HEPES, 20 mM; BSA, 0.1%; and glucose, 0.2%; pH 7.4). The test solution consisted of the standard bath solution and varying concentrations of AVP and Ang II (Sigma Chemical Co., St. Louis, MO), which was applied by puffer superfusion, as previously described (11). This method allowed rapid local solution changes around an identified ZG cell (16). The whole cell Ca; measurement system consisted of a dual wavelength light source (Photon Technology International, Princeton, NJ), an inverted microscope equipped for UV fluorimetry (Nikon Diaphot), and a photon-counting photomultiplier tube (Photon Technology). Fura-2 was used to estimate Cai 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 Kd of 224 nM was used, as previously reported (17). Estimated Caj concentrations fell within a range where fura-2 reliably monitors Ca^
Endo • 1990 Vol 127 • No 2
Identification of ZG cells Cells were identified as ZG cells based on the morphological characteristics described by Tait et al. (18). Cells were spherical, with a diameter of 7-12 jum. The nucleus was round and occupied much of the cell volume, while the cytoplasm contained numerous small lipid droplets. Data analysis Ca; responses for each Ang II and AVP concentration were analyzed by one-way analysis of variance and the NewmanKeuls multiple comparison test. All Ca; recordings were digitally filtered with a 21-point Savitsky-Golay smoothing routine.
Results Individual ZG cells from over 30 different cell preparations were examined. Each ZG cell was stimulated with at least one concentration of either peptide secretagogue, AVP or Ang II. Secretagogue concentrations ranged from 100 pM to 10 ixu for AVP and 50 pM to 10 nM for Ang II; the duration of stimulation was 10-30 min. In most cases, cells were also tested for sensitivity to external K+ with a brief pulse of solution including 10 mM K+. Over 95% of the ZG cells chosen by morphological criteria responded with a rise in Ca; to this elevation in external K+, corroborating their identity as ZG cells. The mean resting Cai level was 98 ± 5 nM (±SEM; n = 239) for responsive ZG cells and was not significantly different for the subpopulations tested at each hormone concentration. Basal Ca; was stable and did not display any oscillatory behavior. Cai response patterns for A VP and Ang II stimulation Responses were substantially different between AVP and Ang II concentrations, as shown in Figs. 1 and 2. With AVP concentrations of 1 and 10 nM, there was a long delay before a sharp Ca; rise. This initial Cai elevation was typically transient, decaying toward basal levels, and was often followed by additional rapid increases. An intermediate concentration of AVP (100 nM) produced an initial Ca; rise with a shorter delay than that found at lower concentrations. The ZG cells showed some variability in the signal pattern; however, a majority of the cells demonstrated multiple oscillations. The peak amplitude of the Ca; oscillations was similar over the period of AVP stimulation in some responsive cells, although there was a tendency for the peak Ca; change to diminish over time in other cells. At the higher concentrations (1 and 10 ixM), the delay in the Ca; response did not vary with concentration, and the initial Caj increase was similar to that found at other AVP concentrations. With these higher doses, the Ca; signal exhibited oscillatory behavior or appeared to fuse into a more sustained Ca; increase. At the lowest dose of AVP tested (100 pM), no
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i RESPONSES OF ADRENAL CELLS TO AVP AND ANG II 500
AVP
400r
10 uM
543 Ang II
500 pM
1 uM
100 pM
100 nM 50 pM
10 nM 5 pM
1 nM
200 400 600 time, seconds FIG. 1. Time course of the Ca; responses to different AVP concentrations. Shown are representative Ca; recordings from five different ZG cells during puffer superfusion with the indicated concentrations of AVP. The duration of stimulation was 10 min, and arrows indicate the onset of secretagogue application. The dashed lines are extensions of the basal Ca; concentration. Similar results were found for 5-14 cells at each concentration.
Ca; changes were observed during a 10-min stimulation (n = 6). Ang II concentrations between 5-500 pM produced Ca; responses similar to those elicited by AVP, including a dose-dependent response delay and Ca; oscillations, especially prominent at lower secretagogue levels (Fig. 2). At concentrations of 500 pM Ang II and above, single ZG cells typically displayed an initial Ca; transient, which decayed to a sustained plateau value (13). Comparison of the Ca; response patterns for AVP and Ang II raises the possibility of a difference in ZG cell sensitivity toward these agonists. Ca; responses to AVP at any given concentration resembled responses to Ang II at doses 2-3 log units lower. Thus, the early Ca; responses to 100 nM AVP were similar to the early Ca; changes produced by 100 pM Ang II.
100 200 300 time, seconds FIG. 2. Time course of the Ca; responses to different Ang II concentrations. Traces are representative Caj recordings from four different ZG cells during puffer superfusion with the indicated concentrations of Ang II. The duration of stimulation was 5 min, and arrows indicate the onset of secretagogue application. The dashed lines are extensions of the basal Cai concentration. Similar results were found for 10-49 cells at each concentration.
Ang II. For example, there was a response delay between secretagogue application and the initial Ca; peak, which is dose dependent. As previously found with Ang II (13), the delay in the response becomes shorter as the AVP concentration is increased, with a mean delay of 209 ± 34 sec at 1 nM and 23 ± 7.5 sec at 1 /*M (Fig. 3). The delay was constant for AVP concentrations of 100 nM and above, indicating saturation of this process. A small number of cells (4 of 79) were not included in the analysis of the response delay, as they exhibited delays in excess of 150 sec with application of 1 /uM and 100 nM AVP. The inverse relationship between hormone concentration and response delay was similar when the cells were stimulated with Ang II, but there was a leftward shift of the dose response. When plotted as a log-log relationship, the results from each hormone become linearized, with equivalent slopes (AVP: -1.176; r = 0.75; Ang II: -1.150; r = 0.88) but different intercepts, indicating an apparent shift in the sensitivity by 2-3 log units. Peak Cai changes with A VP and Ang II
Cai response delay for A VP and Ang II Cas response patterns showed substantial concentration-dependent differences with stimulation by AVP or
The amplitude of the Ca; peak that followed the delay period demonstrated only a modest dose dependency (Fig. 4). The mean peak increase at all AVP concentra-
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RESPONSES OF ADRENAL CELLS TO AVP AND ANG II
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Endo • 1990 Vol 127 • No 2
1
„- 400 ho
-g200
1 1
1
200 T3
100
AVP 10 nM 100 nM 0.1 uM luM 100 pM
Angll 0L
0.01
hormone, nM
100
AVP Ang II FIG. 4. Relationship between peak Caj change and hormone concentration. The peak Cai change is the maximal Cai increase above basal values elicited during a 5- to 10-min AVP or Ang II application. Each data point represents the mean (±SEM) from 5-14 cells for different
AVP concentrations and 10 cells for 100 pM Ang II. The Ang II data were derived from the set of cells which had been stimulated by both hormones (see Fig. 5).
B CO
72200 O
Subpopulations of cells responsive to A VP and Ang II
"100
The maximal steroid stimulation is much lower for AVP than for Ang II (1, 2, 5). This difference could be related in part to the distribution of hormone-responsive ZG cells. This possibility was evaluated by stimulating individual cells with both 100 nM AVP and 100 pM Ang II. Hormone concentrations were chosen that activated the phosphoinositide system to a similar extent (5) but produced little or no heterologous desensitization of the Ca; response (our unpublished observation). Representative records from five different ZG cells, stimulated consecutively with the two peptides, are shown in Fig. 5. The order of presentation of the peptide secretagogues was varied to avoid any systematic error. All cells had robust Ca; responses to 100 pM Ang II; however, only 60% of these cells responded to 100 nM AVP. The AVP and Ang II responses had similar peak amplitudes and rates of Ca; rise, and differences in the response delays could be predicted from the dose-response delay relationship mentioned above. A similar distribution was found for paired stimulations using 10 mM external K+ and a range of AVP concentrations, with over 90% of the ZG cells responsive to external K+, but only 60% showed Caj changes with AVP. Conversely, more than 95% of ZG cells, when challenged with paired stimulations of external K+ and Ang II, elicited Ca; responses to both secretagogues.
"5 50 CO
£ a> 20 10
0.01
1 100 hormone, nM FlG. 3. Relationship between the delay of the Ca; response and hormone concentration. A, The delay of the Cai response is the time elapsed between the start of AVP or Ang II stimulation and the initial rise in Cai. Each data point represents the mean (±SEM) from 5-14 cells for AVP and 10-49 cells for Ang II. B, Response delay vs. hormone concentration plotted in log-log form. The dashed line was fitted to the data points by linear regression for AVP concentrations between 1-100 nM (r = -0.746; P < 0.001) and for Ang II concentrations between 5 pM and 100 nM (r = -0.882; P < 0.001).
tions was 337 ± 51 nM, with a range of 263 ± 52 nM (1 nM AVP) to 407 ± 49 nM (1 /*M AVP). Despite these differences in the mean values, paired comparisons between doses did not reveal any significant differences (P > 0.05). It should be noted that at all AVP concentrations, responsive ZG cells usually produced a number of Ca; transients, with the frequency of these peak changes being a more discriminating feature of the Ca; response than the peak Cai change. As seen in Fig. 2 and reported previously (13), the peak Ca; changes with Ang II also showed little dose dependency. In addition, both secretagogues produced similar Ca; transients when individual cells were exposed to both hormones (Fig. 5).
Early Cat and aldosterone responses to A VP and Ang II As exemplified in Fig. 5, ZG cells typically exhibited oscillatory Ca; behavior at 100 nM AVP; however, single transients were also observed in some cells. In addition, the amplitude of the peak Ca; and/or the frequency of oscillations appeared to decrease over time in many cells
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Ca; RESPONSES OF ADRENAL CELLS TO AVP AND ANG II AVP, 100 nM
Ang II, 100 pM
lOOOr cell 1
cell 2
cell 3
545
nM AVP were mirrored by the aldosterone production pattern. There was a lag time of approximately 4 min between the Ca ; and aldosterone responses, which may be the time required for the steroid to be produced and released in the bath after stimulation by an increased Ca ; or may simply reflect differences in the methods of measurement. The steroid response during Ang II stimulation followed a similar time course for the initial phase; however, aldosterone production continued to rise, while the Ca ; response is maintained. Thus, the kinetics of aldosterone production and mean Ca ; responses correlated at early times. Normalized steroid results show that the full aldosterone response has a different temporal pattern for AVP and Ang II, particularly at early times (Fig. 6C).
Cat and aldosterone response patterns to prolonged A VP and Ang II stimulation cell 4
cell 5
0
300 0 time, seconds
300
FlG. 5. Comparison of Ca; responses from individual ZG cells stimulated by AVP and Ang II. Consecutive stimulations of 100 nM AVP and 100 pM Ang II were presented to individual ZG cells, and the responses of five ZG cells are shown. The order of hormone application was alternated for different ZG cells, but the AVP responses are presented first for simplicity. Arrows indicate the onset of hormone application, and the first 5 min of each Caj response are displayed. Similar results were obtained for a total of 12 ZG cells.
stimulated with AVP (100 nM), while no such phenomenon was apparent when individual cells were stimulated with Ang II (100 pM). To further characterize this decline in the Ca; response, the mean Ca; responses to 100 nM AVP and 100 pM Ang II were determined from individual ZG cell records and plotted vs. time (Fig. 6A). With prolonged AVP stimulation, the Ca; response declined over the first 10 min, reflecting the tendency for the peak Ca; changes and the oscillation frequency to decrease. On the other hand, the Ca; response to Ang II was sustained after the initial increase, just as the oscillatory behavior was maintained for the duration of stimulation. The difference in the peak amplitude of the mean Ca; responses was due to an adjustment made for the percentage of ZG cells responsive to each secretagogue. The aldosterone response pattern shared some of the characteristics of the Ca; signal (Fig. 6B). The sharp rise and gradual decline in the mean Ca; response with 100
Ca; and steroid were measured during prolonged applications of AVP and Ang II to better compare the response patterns to these secretagogues. The aldosterone stimulation by 100 nM AVP was transient and modest. Steroid output rose 5-fold, peaked at 7 min, and returned to basal values within 30 min (Fig. 7). Single cell Ca; records demonstrated a similar time course, as seen in the representative trace in Fig. 7. After the initial set of Ca; oscillations ended, Ca; returned to resting values within 15 min (n = 7). While the Ca; response may persist during the falling phase of aldosterone stimulation, individual ZG cells show a less robust Ca; change, which is reflected in the mean Caj analysis (Fig. 6A). With 100 pM Ang II, the rate-of-rise of aldosterone production was similar to that after AVP stimulation; however, the time required to reach half-maximal stimulation was longer due to the continued increase in steroid production. Peak secretion was reached at 20 min, and aldosterone output remained elevated for the duration of stimulation. The Ca; response to Ang II also showed an oscillatory pattern during the entire 30-min observation period, indicating major differences between the two peptides in the steroid and Ca; responses patterns (n = 10). A 100-pM dose of Ang II produced half-maximal stimulation of aldosterone production by this hormone. Discussion Individual adrenal ZG cells displayed complex changes in Ca; during stimulation with AVP and Ang II. At high concentrations of AVP, an initial rapid rise in Ca; was followed by Ca; oscillations, which appeared to fuse in some cases. At lower AVP concentrations, several changes in the Ca; response were observed, including a lengthening of the response delay and a slowing of the oscillation frequency. Ca; transients or oscillations were
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546
Ca; RESPONSES OF ADRENAL CELLS TO AVP AND ANG II
FIG. 6. Early Cai and aldosterone responses to AVP and Ang II. A, Cai traces represent mean Ca; changes during 10min stimulations by 100 nM AVP and 100 pM Ang II. Cells that did not respond to these doses of hormone were included in calculation of the mean Cai change. The number of responsive cells was 14 of 23 for AVP and 10 of 12 for Ang II. The Ang II data were derived from the set of cells which had been stimulated by both hormones (see Fig. 5). B, Aldosterone responses are the mean values during the first 10 min of stimulation with 100 nM AVP (n = 4) and 100 pM Ang II (n = 5). Effluent fractions were taken every 2-5 min. C, Aldosterone responses were normalized to the mean of the maximal steroid value for each peptide concentration and plotted over time. The maximal aldosterone output was 1.5 ng/ r a t h for 100 nM AVP and 24.7 ng/rath for 100 pM Ang II from a mean basal value of 0.3 ng/rat-h.
Endo• 1990 Vol 127-No 2
2 100
0
5 10 time, minutes
AVP, 100 nM
0
5 10 time, minutes
0
W 1 0 time, minutes
Ang II, 100 pM
FIG. 7. Cai and aldosterone response patterns to prolonged AVP and Ang II stimulation. A, Top, Aldosterone production during a 30-min stimulation with 100 nM AVP from one representative experiment (n = 4). A, Bottom, A representative Ca; response from a single ZG cell during a 30-min stimulation with 100 nM AVP (n = 7). B, Top, Aldosterone production during a 30-min stimulation with 100 pM Ang II from one representative experiment (n = 5). B, Bottom, A representative Ca; response from a single ZG cell during a 30-min stimulation with 100 pM Ang II (n = 10). 10 20 time, minutes
observed in 80% of the AVP-responsive cells, indicating that this was a general response pattern to this peptide. Ca; changes in response to Ang II stimulation shared some characteristics with those stimulated by AVP, but displayed a greater range of response patterns. Stimulation by Ang II in the concentration range from 5-100 pM produced an oscillatory change in Ca;. At Ang II concentrations above 100 pM, ZG cells show an initial Cai transient that decays to a sustained plateau response still substantially above basal levels (12). This response pattern was not seen with AVP stimulation at any dose. In fact, the initial oscillatory response to 100 nM AVP resembled the early Ca; changes produced by 100 pM Ang II, indicating a significantly greater sensitivity for Ang II. The ampltiude of the Ca; transients showed no differ-
10 20 time, minutes
ences between AVP and Ang II and, at best, a modest dose dependency for each secretagogue. While oscillation frequency appeared to decrease with lowering of the hormone concentration, 5- to 10-min stimulations were not sufficiently long to quantitate this parameter. Comparison of the response delays for AVP and Ang II can help clarify how these hormones modify cellular function. The response delay is probably related to the kinetics of receptor binding, the number of receptors per cell, and the efficacy of coupling to effector systems. Both AVP and Ang II elicited a response delay that was inversely related to peptide concentration. When plotted as a log-log relationship, the delay was linearized, revealing an inverse power relationship to hormone concentration with similar slopes for AVP and Ang II. These
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Ca; RESPONSES OF ADRENAL CELLS TO AVP AND ANG II equivalent slopes may be related to the similar binding constants of AVP and Ang II for their respective receptors (1, 19, 20). On the other hand, the shift in the doseresponse curves is probably due to the greater density of Ang II receptors, as reported in previous studies (1, 19, 20). Ins-1,4,5-P3 activation of Ca2+ release is thought to produce the observed Ca; transients, and the rapid generation of Ins-1,4,5-P3 reported for AVP (2) and Ang II (21, 22) is consistent with the delay of the Caj response. These Cai transients had a similar waveform, regardless of the peptide secretagogue (Ang II or AVP) or its concentration, implying a common mechanism for eliciting the initial Caj signal and subsequent oscillations. The modest dose dependency of the peak amplitude may reflect synchronous Ca2+ release within the cell once the release mechanism is activated. These data suggest a threshold for activation of Ca2+ release into the cytosol, which is driven to completion once stimulated. This hypothesis is inconsistent with the graded release produced by Ins-1,4,5-P3 in permeabilized ZG cells (7). On the other hand, a study on neutrophils found positive cooperativity with Ins-1,4,5-P3 activation of Ca2+ release when Ca2+ reuptake was blocked (23). These findings with neutrophils may help to explain both the rapid onset of the Ca; transient and the independence of its amplitude from secretagogue dose. Other positive feedback mechanisms, such as Ca;-dependent Ca2+ release, may also contribute to the Ca; transient (24). In cell population studies, peak Ca; responses have a strong dose dependency, which is commonly correlated with second messenger formation and steroid production (8, 10, 11, 25). However, individual ZG cells as well as other cell types (24, 26) have a remarkably consistent Ca; transient, independent of the stimulus or its concentration. Apparently, it is not the actual magnitude of the Ca; transient that underlies the observed dose dependency of the Cai rise measured in cell populations; rather, this dose dependency results from the extent to which Ca; responses in single cells are synchronized after presentation of the calcium-mobilizing hormone. Therefore, the critical steroidogenic features of the Caj response must reside in the temporal pattern of the Ca, signal found in single ZG cells and the interaction of the Ca; transients with other transduction processes. Differences in maximal steroid stimulation by AVP and Ang II may be related to the distribution of hormoneresponsive ZG cells within the general ZG cell population. Studies using paired AVP and Ang II stimulations of individual cells revealed a subpopulation of AVPresponsive ZG cells. Over 90% of cells stimulated with Ang II responded with a Ca; response; however, only 60% of the Ang Il-responsive cells elicited Ca; changes with AVP. Potential heterologous desensitization was con-
547
trolled by using hormone concentrations that did not produce this phenomenon and by varying the order of stimulation. Similar results were found for paired stimulations of AVP and external K+, while responsiveness to external K+ and Ang II tracked closely together within ZG cells (>95%). The smaller percentage of AVP-sensitive ZG cells can in part explain the reduction in maximal steroid production with AVP stimulation, as there would be fewer cells stimulated by AVP than Ang II. While there are at present no satisfactory means for studying aldosterone production at the single cell level, superfusion measurement of cell population steroid output offers temporal information that can be used to correlate single cell and mean Ca; signals with aldosterone production. The importance of the temporal aspects of the Ca; signals is illustrated in the comparison of steroid responses to AVP and Ang II, both of which involve the phosphoinostide system. When using AVP (100 nM) and Ang II (100 pM) concentrations that produce similar activation of the phosphoinositide system (5) and early Cai responses, aldosterone production induced by AVP rose sharply, with a delay and rate of rise equivalent to those found with Ang II stimulation. However, single cell Ca; declined to basal levels within 15 min, with no additional Ca; increases observed in the next 15 min of AVP stimulation. This termination of the Ca, response may explain the decline of steroid secretion to basal levels found with AVP stimulation. For Ang II, the Cai and steroid responses were maintained for the duration of hormone application. Interestingly, despite the dramatically different time courses for the Ca; responses to AVP and Ang II, both peptides produce sustained activation of the phosphoinositide system (5). Active and inactive inositol trisphosphate isomers were not separated in this study; therefore, it is possible that these hormones stimulate alternate metabolic pathways for Ins-1,4,5-P3. This could lead to the production of different sets of inositol polyphopsphates, which could alter the regulation of Ca;. Previous studies comparing steroid responses to these calcium-mobilizing hormones found substantially slower aldosterone production in response to 100 pM Ang II than in response to 100 nM AVP, while the maximal steroid outputs were similar (1, 5). When steroid data from the present study were normalized, the half-time for maximal aldosterone stimulation by Ang II was also substantially slower than that of AVP. Likewise, the peak of the aldosterone response was later for lower Ang II concentrations that stimulated the cells to similar maximal steroid levels as did 100 nM AVP (our unpublished data). Therefore, these differences from previous studies may simply be due to a greater sensitivity of the ZG cell to Ang II in preparations used in the present study.
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548
RESPONSES OF ADRENAL CELLS TO AVP AND ANG II
It is impossible at this time to determine whether stimulation by calcium-mobilizing hormones involves frequency modulation of steroid production or a simple integration of the Ca; signal. Ca; oscillations are part of the transduction mechanism for AVP and Ang II and may have important effects on Ca2+-dependent processes through the generation of large localized Ca; changes near the Ca2+ source; however, the most effective Ca2+ signals for activation of the steroidogenic pathways are not well understood. Given the differences in the Ca; response patterns to the secretagogues AVP, Ang II, and K+, closer examination of the relationship between changes in Ca; and aldosterone may provide additional insights into the cellular roles of these Ca; signals. Acknowledgments Expert technical assistance was provided by Alpheen Menachery, Tham Yao, and Paul Lartouris.
References 1. Balla T, Enyedi P, Spat A, Antoni FA 1985 Pressor-type vasopressin receptors in the adrenal cortex: properties of binding, effects on phosphoinositide metabolism and aldosterone secretion. Endocrinology 117:421 2. Woodcock EA, McLeod JK, Johnston CI 1986 Vasopressin stimulates phosphatidylinositol turnover and aldosterone synthesis in rat glomerulosa cells: comparision with angiotensin II. Endocrinology 118:2432 3. Guillon G, Gallo-Payet N 1986 Specific vasopressin binding to rat adrenal glomerulosa cells: relationship to inositol lipid breakdown. Biochem J 235:209 4. Gallo-Payet N, Guillon G, Balestre MN, Jard S 1986 Vasopressin induces breakdown of membrane phosphoinositides in adrenal glomerulosa and fasciculata cells. Endocrinology 119:1042 5. Enyedi P, Balla T, Antoni FA, Spat A 1988 Effect of angiotensin II and arginine vasopressin on aldosterone production and phosphoinositide turnover in rat adrenal glomerulosa cells: a comparative study. J Mol Endocrinol 1:117 6. 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 7. Rossier MF, Krause K, Lew PD, Capponi AM, Vallotton MB 1987 Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. J Biol Chem 262:4053 8. Braley L, Menachery A, Brown E, Williams G 1984 The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells. Biochem Biophys Res Commun 123:810
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9. Capponi AM, Lew PD, Jornot L, Vallotton MB 1984 Correlation between cytosolic free Ca++ and aldosterone production in bovine adrenal glomerulosa cells. J Biol Chem 259:8863 10. Kramer RE 1988 Angiotensin II causes sustained elevations in cytosolic calcium in glomerulosa cells. Am J Physiol 255:E338 11. Balla T, Hausdorff WP, Baukal AJ, Catt KJ 1989 Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin II. Arch Biochem Biophys 270:398 12. Connor JA, Cornwall MC, Williams GH 1987 Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques. J Biol Chem 262:2919 13. Quinn SJ, Williams GH, Tillotson DL 1988 Calcium oscillations in single adrenal glomerulosa cells stimulated by angiotensin II. Proc Natl Acad Sci USA 85:5754 14. Williams GH, McDonnell LM, Raux MC, Hollenberg NK 1974 Evidence for different angiotensin II receptors in rat adrenal glomerulosa and rabbit vascular smooth muscle cells. Circ Res 36:384 15. Enyedi P, Szabo B, Spat A 1985 Reduced responsiveness of glomerulosa cells after prolonged stimulation with angiotensin II. Am J Physiol 248:E209 16. Quinn SJ, Williams GH, Tillotson DL 1988 The calcium response to external potassium in single adrenal glomerulosa cells Am J Physiol 255:E488 17. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440 18. Tait JF, Tait SAS, Gould RP, Mee RSR 1974 The properties of adrenal zona glomerulosa cells after purification by gravitational sedimentation. Proc R Soc Lond [Biol] 185:375 19. Glossman H, Baukal AJ, Catt KJ 1974 Properties of angiotensin II receptors in the bovine and rat adrenal cortex. J Biol Chem 249:825 20. Douglas J, Aguilera G, Kondo T, Catt K 1978 Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells. Endocrinology 102:685 21. Enyedi P, Buki B, Mucsi I, Spat A 1985 Polyphosphoinositide metabolism in adrenal glomerulosa cells. Mol Cell Endocrinol 41:105 22. Balla T, Baukal AJ, Guillemette G, Morgan RO, Catt KJ 1986 Angiotensin-stimulated production of inositol triphosphate isomers and rapid metabolism through inositol 4-monophosphate in adrenal glomerulosa cells. Proc Natl Acad Sci USA 83:9323 23. Meyer T, Holowka D, Stryer L 1988 Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate. Science 240:653 24. Berridge MJ, Cobbold PH, Cutherbertson KSR 1988 Spatial and temporal aspects of cell signalling. Phil Trans R Soc Lond [Biol] 320:325 25. Capponi AM, Lew PD, Vallotton MB 1987 Quantitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine adrenal glomrulosa cells: different requirements for angiotensin II and K+. Biochem J 247:355 26. Berridge MJ, Galione A 1988 Cytosolic calcium oscillators. FASEB J 2:3074
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