Angiotensin II-stimulated changes m calcium metabolism in cultured glomerulosa cells

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Molecular and Cellular Endocrinology, 60 (1988) 199-210 Elsevier Scientific Publishers Ireland, Ltd. MCE 01956

Angiotensin

II-stimulated changes in calcium metabolism in cultured glomerulosa cells Robert

Department

E. Kramer

ofPharmacology, Univemity of Tennessee, Memphis, The Health Science Center, Memphis, TN 38163, U.S.A. (Received 2 June 1988; accepted 1 August 1988)

lvey words: Cytosolic free calcium: Fura 2: Calcium methoxybenzoate (TMB-8)

influx;

Calcium

efflux;

Dantrolene;

S-(~,~~-~ethyl~no)-octyl-3,4,~-t~-

Studies were performed to evaluate the relations~ps between the effects of angiotensin II on calcium metabolism and cytosolic free calcium concentration in primary monolayer cultures of bovine adrenal glomerulosa cells. As noted previously (Kramer (1988) Am. J. Physiol. (in press)), angiotensin II produced rapid dose-dependent increases in cytosolic calcium characterized by both an initial transient component and a secondary sustained component. In the absence of extracellular calcium, angiotensin II produced an initial increase in cytosolic calcium comparable to that produced in the presence of calcium, but failed to maintain a sustained calcium signal. The initial, angiotensin-stimulated increase in cytosolic calcium was inhibited by dantrolene and 8-( N, N-diethylamino)-octyL3,4,5trimethoxybenzoate (TMB-8) in a concentration-dependent fashion. The onset of the angiotensin-stimulated calcium signal was accompanied by a dose-dependent increase in the rate of calcium efflux that achieved a maximum within 2-3 min and then declined to a level 2.5-3 times that from control cells. The initial rate of calcium influx was also increased about 2.5-fold by angiotensin II, an effect that was only apparent in cells that had been treated with the peptide for at least 5 min. These results indicate that the calcium signal produced by angiotensin II is initiated by the rapid mobilization of calcium from an intracellular site(s) and sustained by the continued uptake of extracellular calcium. Moreover, the kinetics of the calcium signal as well as the final, sustained calcium concentration achieved reflect the balance between intracellular calcium release, calcium influx and calcium efflux.

Address for correspondence: Robert E. Kramer, University of Tennessee, Memphis, Department of Pha~acology, 100 Crowe Building, 874 Union Avenue, Memphis, TN 38163, U.S.A. Abbreviations: AII, angiotensin II; BHA, butylated hydroxyanisole; BSA, bovine serum albumin; [Ca2+ I,, cytosolic free calcium concentration; DMSO, dimethylsulfoxide; DNase, deoxyribonuclease; HBSS, Hanks’ balanced salt solution; Hepes, 4-(Z-hydroxyethyl).l-piperazineethanesulfonic acid; IP,, inositol trisphosphate; TMB-8, S-( N, N-diethylamino)-octyl3,4,5_trimethoxybenzoate. 0303-7207/88/$03.50

Introduction The importance of calcium in the action of angiotensin II to increase aldosterone production by the adrenal glomerulosa cell is well established (Fakunding et al., 1979; Fakunding and Catt, 1980; Foster et al., 1981; Sehiffrin et al., 19SI). Binding of angiotensin II to specific receptors on the surface of the glomerulosa ceI1 is thought to initiate a series of biochemical events that in-

0 1988 Elsevier Scientific Publishers Ireland, Ltd.

fluences the mobilization of calcium from an intracellular site, presumably the endoplasmic reticulum (Farese et al., 1984; Kojima et al., 1984; Enyedi et al., 1985) as well as the movement of calcium across the plasma membrane (Elliot and Goodfriend, 1981; Foster and Rasmussen, 1983; Elliott et al., 1985; Kojima et al., 1985a, b, c). One of the i~ediate consequences of the effects of angiotensin II on calcium mobilization appears to be an accumulation of calcium within the cytosol of the glomerulosa cell (Braley et al., 1984; Capponi et al., 1984; Kojima and Ogata, 1986; Kramer, 1988). Moreover, it has been generally accepted that the increase in cytosolic free calcium concentration [Ca’+],, produced by the peptide is essential for the stimulation of aldosterone biosynthesis and secretion. The relationships between the effects of angiotensin II on calcium dynamics, [Ca2’-1, and aldosterone output, however, are not completely understood. For example, angiotensin II produces a sustained increase in aldosterone formation that is attenuated by calcium channel antagonists (Fakunding et al., 1979; Fakunding and Catt, 1980; Schiffrin et al., 1981). Attempts to demonstrate directly an increase in calcium uptake, however, have yielded conflicting results (Elliott and Goodfriend, 1981; Foster and Rasmussen, 1983; Elliott et al., 1985; Kojima et al., 1985a, b). In addition, extracellular calcium comprised a minor component of the calcium signal produced by the peptide in isolated bovine glomerulosa cells (Capponi et al., 1984), although it contributed significantly to that in the isolated rat glomerulosa cell (Braley et al., 1986). Moreover, angiotensin II produced only a transient increase in [Ca2’], fcappo~rii et- a%-;-l984; #oj?ma and -Ogafa;- 1935); leaving the means by which changes in calcium metabolism would be transduced into a sustained steroidogenic response unclear. In contrast to studies using acutely dispersed glomerulosa cells, recent studies performed in this laboratory using bovine glomerulosa cells maintained in primary culture demonstrated clearly that angiotensin II produced a sustained increase in [Ca2+], (Kramer, 1988). These results suggest that an elevation in [Ca2+], is more important in the maintenance of angiotensin-stimulated aldosterone secretion than heretofore proposed. Re-

cent studies using cultured mesengial (Hassid et al., 1986) and vascular smooth muscle (Brock et al., 1985; Nabika et al., 1985) cells have also indicated that angiotensin II caused a sustained increase in [Ca2+],. In cultured vascular smooth muscle cells (Nabika et al., 1985) the nature of the calcium signal produced by angiotensin II was markedly dependent upon the c~nce~trat~o~ of the peptide, an effect that apparently reflected dose-dependent differences in the mechanism(s) by which angiotensin II increased [Ca2’],. The characteristics of the angiotensin-stimulated calcium signal in cultured adrenal glomerulosa cells were also dependent (Kramer, 1988), but it is presently unknown whether or not dose-dependent effects of angiotensin II on calcium mobilization might underlie those in [Ca”],. Therefore, the present studies were performed to investigate further the effects of angiotensin II on calcium metabolism in bovine glomerulosa cells maintained in primary monolayer culture and to define their relationships to changes in [Ca2’],. The results reported here support previous observations that angiotensin II produces a sustained increase in [Ca2+],. Furthermore, they indicate that the calcium signal produced by angiotensin II consists of an initial, transient component resulting from the rapid mobilization of an intracellular calcium pool(s) and a secondary, sustained component dependent upon the uptake of extracellular calcium. The dose-dependent characteristics of the angiotensin XI-stimulated calcium signal appear to reflect the integration of those on the mobilization of intracellular calcium, calcium influx and calcium efflux. Materials and methods Cell culture Glomerulosa cells were isolated by collagenase/ hyaluronidase digestion and mechanical dispersion of capsular tissue from lo-12 adrenal glands collected from freshly slaughtered heifers and established in primary culture as previously described (Kramer, 1988). Cells were seeded to 16 mm x 93 mm leighton tissue culture tubes containing a 9 mm X 35 mm glass coverslip or fibronectin-coated (2 pg/cm2) 6 well-cluster (35

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mm) dishes in a modified Ham’s F-12/Hepes (25 mM) medium containing antibiotics/antimycotics and 10% horse serum. Cells were maintained at 37°C in a humidified atmosphere of nitrogen (90%) supplemented with air (10%) flowing at a rate of 1 liter/mm. Medium was replaced at 48 h intervals. Confluence was generally achieved within 5-7 days. At confluence, cell monolayers were incubated for an additional 24-48 h in F-12 medium supplemented with protease-free BSA (200 pg/ml), but in the absence of serum, insulin, and transferrin. Ham’s F-12 medium was obtained from Whittaker M.A. Bioproducts (Walkersville, MD). HBSS, serum and antibiotic/antimycotic solutions were obtained from Grand Island Biological Company (Grand Island, NY). Tissue culture dishes were purchased from Costar (Cambridge, MA). Other plastic labware, leighton tubes and coverslips were from Bellco Biotechnology (Vineland, NJ). Collagenase (type II) was from Worthington (Freehold, NJ). Protease-free BSA was obtained from Boehringer Mannheim (Indianapolis, IN). Fibronectin and other components of the cell culture and digestion media were obtained from Sigma Chemical Co. (St. Louis, MO). Cyfosoliccalcium The concentration of free calcium in the cytosol of adherent glomerulosa cells was measured using the fluorescent calcium indicator fura 2 (Grynkiewicz et al., 1985). Cells attached to a 9 X 35 mm coverslip were incubated for 45 min at 37 o C in 2 ml Ham’s F-12 medium containing Hepes (25 mM, pH 7.4) and fura 2 acetoxymethyl ester (2 PM) added in DMSO (2 pi/ml). Loaded cells were inserted into a 1 ml fluorescence cuvette contained in a Perkin-Elmer LS-5 spectrofluorometer fitted with a water-jacketed cuvette holder maintained at 37” C and superfused at a rate of about 3 ml per minute with HBSS/Hepes (25 mM, pH 7.4) medium contai~ng protease-free BSA (200 pg/ml) and 1.8 mM CaCI, (Hassid et al., 1986; Kramer, 1988). Fluorescence at 510 nm was continuously recorded as the excitation wavelength was alternated at 6 s intervals between 340 nm and 380 nm resulting in a discontinuous recording of fluorescence at each excitation wavelength. Fluorescence at each wavelength was cor-

rected for cell autofluorescence, and the ratio of net fluorescences used to calculate [Ca2’], (Grynkiewicz et al., 1985) using a nM dissociation constant of 224. Calibration parameters were determined as described by Tsien et al. (1985). Preliminary studies indicated that cultured glomerulosa cells readily converted the fura 2 acetoxymethyl ester to the free acid. Essentially all of the fura 2 extracted from loaded cells and subjected to HPLC comigrated with free acid with little, if any, comigrating with the esterified form of the probe. In addition, the maximal fluorescence ratio obtained in fura 2-loaded cells following treatment with ionophore was within 5% of that of the free acid, suggesting that the presence of calcium-insensitive forms of the dye did not significantly contribute to the results of the present investigations (Scanlon et al., 1987). Extracellular fura 2 also appeared to make little cont~bution to the values determined for cytosolic free calcium concentration since the fluorescence of effluent from fura 2-loaded cells was not significantly above background. In addition, Mn2’ (0.1 mM) failed to significantly affect the fluorescence of fura 2-loaded cells (Kramer, 198X). Generally, a relatively constant fluorescence ratio was observed after lo-15 min of superfusion. Thereafter, superfusion was allowed to continue for an additional lo-15 min before an effector was added to the superfusate. Solutions containing the appropriate concentration(s) of effector were prepared at the beginning of an experiment, kept at O--4” C in siliconized glass reservoirs, and warmed to 37°C immediately before use. Dantrolene was the only effector to influence autofluorescence, and appropriate corrections were made for the quantitation of [Ca*+],. Results of preliminary experiments also indicated that essentially all of the fura 2 acetoxymethyl ester taken up by the cells was converted to the non-esterified form of the probe (data not shown). The acetoxymethyl ester and potassium salt of fura 2 were obtained from Molecular Probes (Eugene, OR). Angiotensin II (AH, [ 5Val]AII) was purchased from Bachem (Torrence, CA). TMB-8 was obtained from Aldrich Chemical Company (Milwaukee, WI), dantrolene from Norwich Eaton Pharmaceuticals (Norwich, NY).

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In order to measure calcium efflux, cells attached to a 9 x 35 mm coverslip were incubated for 2 h in 2 ml protein-free Ham’s F-12 medium (1.8 mM CaCl,) containing 10 PCi of [45Ca2f]CaCl,. In experiments in which [Ca2.‘], and efflux of [45Ca2+J were measured simult~eously, fura 2 acetoxymethyl ester (2 t*_M) was added at the end of the first hour. Cells were then subjected to superfusion as described above, and effluent was collected at 1 min intervals. Angiotensin II (1O-1’ M or lo- 9 M) was added to the superfusate after 15 min. Superfusion was stopped after 32 min, and the coverslip removed and immersed in 5 ml of Bio-Safe II (Research Products International, Mount Prospect, IL). Superfusion was then continued for another 3 min to ensure recovery of [45Ca2+] from the tubing located between the cuvette and the fraction collector. The amount of isotopic calcium lost from the cells during each time interval and residual intracellular [ 45Ca*+ ] were determined by liquid scintillation counting. Calcium efflux during each 1 min period is expressed as a percent of the total [45Ca2+] contained within the cells at the beginning of that interval, i.e., the calcium efflux coefficient (Borle et al., 1982). Calcium influx was measured using cell monolayers maintained in 35 mm dishes. Cells were rinsed twice with calcium-free/Hepes (20 mM) buffer (pH 7.4) containing NaCl (120 mM), KC1 (5 mM), MgCl, (1 mM), glucose (10 mM), and EGTA (0.5 mM), and then incubated at 37 o C in 1 ml of the same buffer in the absence (control) or presence (lO_” M or lo-’ M) of angiotensin II for 2, 5, or 15 min. At the times indicated, medium was rapidly aspirated and replaced with 1 ml of calcium-replete (1.2 mM) buffer containing 2 pCi of [ 45Ca2+]CaC1,. The incubation was allowed to continue for up to 2 min and then terminated by the addition of 5 ml cold (O-4 o C) Hepes (10 mM) buffer containing MgCl, (100 mM) and LaCl, (10 mM). Zero time blanks were obtained by adding 5 ml cold magnesium/lanthanum solution to the cells before the addition of [45Ca2+]CaC1,. Monolayers were kept at O-4 o C and rinsed 8 times with 5 ml cold magnesium/ lanthanum/ Hepes buffer. Cells were lysed in 2 ml of 0.1 M nitric acid, and [ 45Ca2’] in the cell lysates measured by liquid

scintillation counting. Residual acid fixed protein in each culture well was rinsed 3 times with 5 ml cold 1 M NaCl, dissolved in 0.5 N NaOH and quantitated by the method of Bradford (1976). Statistical ana@ses Statistical analyses were by analysis of variance with appropriate a posteriori tests. Results Effects of angiotensin II on cytosolic calcium concentration The effects of angiotensin II on [Ca2’], in primary cultures of bovine glomerulosa cells are presented in Fig. 1. [CaZilc increased gradually from a basal level of 61 rt: 9 nM (mean 1: SEM) to 174* If: 8 nM ( *P c 0.01 versus control) in response to lo- l1 M angiotensin II. A maximal increase in [Ca’+], was achieved within 5 min of the onset of superfusion with the peptide and was maintained in the continued presence of angiotensin II. A higher concentration (lo-’ M) of angiotensin II produced a more rapid and intense response with [Ca2’], reaching a maximum of

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Fig. 1. Effects of angiotensin II on cytosolic free calcium concentration. Confluent monolayers of adherent bovine glomerulosa cells were loaded with fura 2, and cytosolic free calcium concentration was assayed by discontinuous dualwavelength fluorescence spectroscopy as described in Materials and Methods. Cells were superfused with a buffer containing 1.8 mM CaCt,, but without agonist, until a relatively constant fluorescence ratio was observed throughout a 10 min period. Superfusion was then continued for another 15 min in the absence (control) or presence (10-i’ M or 10K9 M) of angiotensin II (AII). Values represent the meaniSEM of six determinations on individual monolayers.

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610 * + 8 nM within seconds following exposure of the cells to the peptide. Thereafter, the concentration of calcium in the cytosol rapidly declined, but plateaued at a level (X3* f 7 nM) that was significantly greater than that in control cells. In cells superfused in the absence of angiotensin II [Ca2+], remained relatively constant ranging from 60 + 7 nM at the onset to 69 + 12 nM at the end of superfusion. These findings are consistent with previous observations from this laboratory indicating that angiotensin II produced dose-dependent and sustained increases in [Ca2’], in cultured bovine glomerulosa cells (Kramer, 1988).

Contribution of extracelhlar and intracellular calcium pools to the angiotensin-stimulated calcium signal Results of previous studies (Nabika et al., 1985) using cultured vascular smooth muscle cells suggested that the differences in the kinetics of the calcium signals shown in Fig. 1 may reflect differences in the sources of calcium contributing to the responses to low (IO-” M) and high (low9 M) concentrations of angiotensin II. Thus, experiments were performed to assess the importance of intracellular and extracellular pools of calcium to angiotensin-stimulated changes in [Ca’“], in cultured glomerulosa cells. As expected, in the presence of extracellular calcium (1.8 mM) angiotensin II (lo-10 M) produced a rapid and sustained elevation in [Ca’+], (Fig. 2A). Removal of extracellular calcium (i.e., superfusion with calciumfree buffer supplemented with 0.5 mM EGTA) alone slightly decreased [Ca’+], from 60 & 3 nM to 51 + 1 nM (P < 0.01) (Fig. 2B). In the absence of extracellular calcium, angiotensin II (10-i’ M) initiated an increase in [Cazflc comparable to that initiated in the presence of extracellular calcium (425 + 19 nM vs. 400 + 9 nM), but failed to maintained a sustained calcium signal (compare Fig. 2A and 2B). Upon repletion of calcium (1.8 mM) to the superfusate, [Ca2’}, increased to a level similar to that achieved in response to angiotensin II in the continued presence of extracellular calcium (271 _t 15 nM vs. 263 f 5 nM) (compare Fig. 2A and 2B). In control cells superfused with a calcium-free/EGTA buffer, [Ca2*], returned to normal basal levels following calcium repletion

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Fig. 2. Angiotensin II-stimulated calcium signals in the presence and absence of extracellular calcium. Adherent, glomerulosa cell monolayers were loaded with fura 2, and cytosolic free calcium concentration measured as described in Materials and Methods. In the protocol presented in panel A, cells were first superfused with a buffer containing 1.X mM CaCl, and then with the same buffer containing 10-r” M angiotensin II. Alternatively. cell monolayers were superfused sequentially with buffer containing 1.8 mM CaCl,, calcium-free buffer containing 0.5 mM EGTA, calcium-free/EGTA buffer containing 10-t’ M AI1 and, finally, calcium-replete (1.8 mM) buffer containing 1OK” M AI1 (panel B). In a third protocol (panel C), cells were superfused first with calcium-replete buffer, then calcium-replete buffer containing 10mto M AI1 and, finally, calcium-free/EGTA buffer containing lo-” M AII. Changes in the composition of the superfusate were made as indicated. Values represent the mean+SEM of five determinations using individual monolayers in three separate experiments.

(data not shown). As shown in Fig. 2C, the sustained increase in [Ca2’], established in response to angiotensin II in the presence of extracellular

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Fig. 3. Relationshjp of extracellular calcium to initial and sustained angiotensin If-stimulated changes in cytosolic free calcium concentration. Solid bars represent the initial (panel A) and sustained (panel B) changes in cytosolic free calcium concentration produced by angiotensin II (IO-‘* M-low9 M) in the presence of extracellular calcium, the open bars those caused by the peptide in the absence of extracellular calcium. See Eiig. 2 (panels A and B) for details of the superfusion. The magnitude of the initial calcium signal was determined by the difference between cytosolic calcium concentration at the time of addition of angiotensin II and the maximum concentration achieved during the first 5 mm of superfusion with the peptide. The magnitude of the sustained signal reflects the difference between cytosolic calcium concentrations at the time of addition of angiotensin II and 15 min thereafter. Values represent the mean $: SEM of 4-6 determinations in three separate experiments. *P c: 0.01 versus the response to the same concentration of angiotensin II in the presence of extracellular calcium.

calcium was readily reversed by the subsequent removal of extracellular calcium. The consequence of removing extracellular calcium was not dependent upon the concentration of angiotensin II. As noted in Fig. 3, concentrations of angiotensin II ranging from lo-l2 M to lop9 M, although initiating a calcium signal equivalent in magnitude to that initiated in the presence of extracellular calcium (Fig. 3A), failed to sustain an elevated [Ca2’], in the absence of extracellular calcium (Fig. 3B). As suggested by the data presented in Fig. 38, the duration of the transient calcium signal produced by an~otensin

II in the absence of extracellular calcium was inversely related to the concentration of the peptide. For example, the increase in [Ca2’], produced by lop9 M angiotensin II rapidly decayed to control levels within 5 min when calcium was depleted from the superfusate. The calcium signal produced by lo- lo M angiotensin II, on the other hand, declined to control levels within lo-15 min, and that produced by lo-” M angiotensin II gradually decayed over a period of 15-20 min. Thus, when measured 15 min after the onset of superfusion with angiotensin II in a calciumfree/EGTA supplemented buffer, [Ca”], in cells exposed to 10-i’ M angiotensin II or 10d9 M angiotensin II was slightly increased or decreased, respectively, compared to basal [Ca2+], (Fig. 3B). Similarly, the calcium signal produced by a lower concentration (lo-l2 M) of angiotensin II in the absence of extracellular calcium declined only about 40% after 15 min (Fig. 3B), but completely returned to control levels by approximately 30 min (data not shown). .Effects of dantrolene and TMB-8 on the initial component of the angiotensin-stimulated calcizcm signal Dantrolene and TMB-8 are presumed to act by preventing the mobilization of intracellular calcium (Putney and Bianchi, 1974; Chiou and Malagodi, 1975; Van Winkle, 1976; Clapper and Lee, 1985). Nonetheless, in order to avoid possible effects of the antagonists, particularly those of TMB-8 (Kojima et al., 1986), on calcium influx, the effects of dantrolene and TMB-8 on the magnitude of the initial component of the angiotensin II-stimulated calcium signal were examined in the absence of extracellular calcium. Cells were initially superfused with calcium-replete (1.8 mM) buffer until a stable basal [Ca2’], was observed. Superfusion with calcium-free/O.5 mM EGTA buffer was then begun. After 5 min, vehicle (DMSO, 0.5 ,ul per ml) or antagonist (lo-100 PM) was added to the superfusate, and superfusion continued for another 5 min before addition of angiotensin II (lo-” M). As shown in Fig. 4, both dantrolene and TMB-8 inhibited the initial component of the calcium signal produced by angiotensin II in a concentration-dependent fashion. The IC,, for dantrolene in~bition of the initial,

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Fig. 4. Inhibition of the initial angiotensin II-stimulated calcium signal by dantrolene and TMB-8. The initial calcium signal was assayed essentially as described for Figs. 2 and 3 except that superfusion with calcium-free/EGTA buffer was begun 10 min before the addition of angiotensin II (1OU” M) to the superfusates. Vehicle (DMSO, control) or antagonist (10, 25, 50 or 100 PM) was added to the superfusates 5 min prior to the addition of the peptide. Values for TMB-8 represent the mean + SEM of four determinations using individual monolayers in those for dantrolene three two different experiments, determinations in two separate experiments. All values are expressed as a percent of the control response.

angiotensin-stimulated elevation of cytosolic calcium was approximately 20 PM, that for TMB-8 approximately 50 PM. Effects of angiotensin II on calcium eff2ux Angiotensin II produced rapid and sustained increases in [45Ca] efflux (Fig. 5). By 2 min after the addition of lop9 M angiotensin II to the superfusate, calcium efflux increased from a value of 3.5 + l.O%/min to a maximum of 20.4* f 0.9%/min. The rate of calcium efflux decayed rapidly over the next several minutes but remained at a level roughly 3 times that of control cells (5.2* & 1.0 vs. 1.8 f O.S%/min) after 15 min. The rate of calcium efflux was also increased significantly, albeit to a lesser extent, by a lower concentration (lo- ” M) of angiotensin II. The rate of calcium efflux increased from 3.7 + O.l%/min to 8.3* k 0.6%/min by 3 min after exposure of the cells to lo-” M angiotensin II and then gradually declined. Still, after 15 min the rate of calcium efflux from cells superfused with lo-” M angiotensin II remained at a level (4.8* f 0.8%/min) approximately 2.5 times that from control cells.

The temporal relationships between calcium efflux and [Ca”], measured simultaneously are presented in Fig. 6. As expected, the increase in calcium efflux caused by angiotensin II was closely associated with the onset of the calcium signal. The marked increase in calcium efflux produced by 10V9 M angiotensin II was preceded by that in [Ca”], (Fig. 6A). Calcium efflux produced by 10-i’ M angiotensin II, on the other hand, attained a maximum prior to [Ca2’], (Fig. 6B). In general,. the temporal changes in calcium efflux elicited by angiotensin II closely paralleled the kinetics of the initial, intracellular calcium-dependent component of the calcium signal. Angiotensin-stimulated calcium efflux was abolished by 10 mM lanthanum chloride (Fig. 7). In addition, lanthanum prevented the secondary decline in [Ca2+], characteristic of the calcium signal produced by 10-i’ M angiotensin II (compare Figs. 2A and 7). Effects of angiotensin II on calcium injlux Preliminary experiments indicated that incubation in a calcium-deplete medium, while having no apparent effect on calcium uptake by control cells,

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Fig. 5. Effect of angiotensin II on calcium efflux. Adherent glomerulosa cells were incubated with 10 pCi [ 45CaZ+ ]CaCl, for 2 h, and efflux of isotopic calcium assayed as described in Materials and Methods. Angiotensin II was added to the superfusate at a final concentration of lo-” M or 10m9 M. Control cells were superfused in the continued absence of agonist. Values represent the mean+SEM of four determinaa tions using individual monolayers in two separate experiments. Time courses for angiotensin-invoked changes in calcium efflux have been corrected for the delay required for the superfusate to traverse the distance from the cuvette housing the cells to the collection vial.

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TIME (minutes) Fig. 6. Relationship between angiotensin II-stimulated changes in cytosolic calcium concentration and calcium efflux. Cells were incubated in the presence of [45Ca21]CaC12 (10 PCi) for 2 h with fura 2 acetoxymethyl ester (2 (LM) present during the second hour. Cetls were then superfused with buffer containing and cytosolic calcium concentration and 1.8 mM CaCi,, [ 45Ca2 ’ ] efflux monitored simultaneously. Angiotensin II was added to a final concentration of 10K9 M (panel A) or 1OU” M (panel B). Time courses for angiotensin-stimulated changes in calcium efflux have been corrected for the delay between the fluorescence cuvette and the fraction collector. No corrections have been made for the delay of appro~mately 1 min required for the agonist to traverse the inlet tubing between the buffer reservoir and the cuvette. Values represent the mean & SEM of four determinations on individual monolayers from three separate experiments.

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Fig. 7. Inhibition of angiotensin-stimulated calcium efflux by lanthanum chloride. Cytosolic calcium concentration and 14’Ca2+ ] efflux were measured simultaneously as described in Materials and Methods. See Fig. 6 for additional details. Lanthanum chloride (10 mM) and angiotensin II (1OK” M) were added at the times indicated. Data represent the resutts from a single experiment, The experiment was performed twice using two separate cell preparations with comparable results.

mdicative of calcium influx, was increased 4sCa2+, . by angiotensin II only during the 10 s immediately following addition of [ 45Ca]CaCl,. Thereafter, the amounts as well as the rates of accumulation of

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augmented initial calcium uptake in angiotensintreated cells (data not shown). Therefore, in order to enhance the measurement of angiotensinstimulated calcium uptake, glomerulosa cell monolayers were incubated in a calcium-free/O.5 mM EGTA buffer with or without angiotensin II for up to 15 min. Then, medium was replaced with a calcium-replete (1.2 mM) buffer containing tracer amounts of ]45Ca]CaC1,, and calcium uptake assayed over the next 2 min. Time courses for the accumulation of 45Ca2’ in cells preincubated for 15 min in calcium-free/EGTA buffer in the absence (control) or presence of lop9 M angiotensin II (AII) are presented in Fig. 8. Intracellular

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TIME (seconds) Fig. 8. Acc~ulation of 4sCa2+ in monolayer cultures of bovine glomerulosa cells. Monolayers were incubated in the absence (control) or presence of angiotensin II (AH; 10m9 M) for 15 min in calcium-free/O.5 mM EGTA buffer. Calcium-deplete buffer was then rapidly replaced with 1 ml calcium-replete (1.2 mM) buffer containing 2 pCi [45Ca2+]CaC12. The incubation was terminated at the times indicated by the addition of cold ma~esi~/l~th~um (100 mM/lO mM) buffer. Values represent the meant SEM of nine determinations in three separate experiments. *P < 0.01 versus control.

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Fig. 9. Effect of angiotensin II on initial calcium uptake. Cells were incubated for up to 15 min in a calcium-free/O.5 mM EGTA buffer in the absence (control) or presence of angiotensin II (AIL 10m9 M). At the times indicated, calcium-deplete buffer was rapidly replaced with 1 ml of buffer containing 1.2 and 2 pCi [45Ca2f]CaC1,. Incubations were mM C&l, terminated 10 s later by the rapid addition of cold buffer containing 100 mM MgCl, and 10 mM LaCl,. Values represent the mean+ SEM of six determinations in two different experiments. *P -c 0.01 versus control.

45Ca2t in control and angiotensin-treated cells were not significantly different (Fig. 8). As noted in Fig. 9, the effect of angiotensin II to increase the initial (O-10 s) rate of calcium uptake was not immediately apparent. Initial calcium uptake by cells exposed to lop9 M angiotensin II for up to 2 min was indistinguishable from that by control cells. An increase in initial calcium uptake, however, was apparent in cells preincubated with angiotensin II for 5 and 15 min (Fig. 9). Initial calcium uptake in cells treated for 15 min with lO_” M angiotensin II was similar to that of cells treated with 10m9 M angiotensin II (47.5* + 3.0 and 46.1* f 3.9 pmol/lO s/mg protein, respectively, compared to 26.4 k 2.6 pmol/lO s/mg protein in control cells). Discussion The present studies were performed to evaluate the changes in calcium metabolism underlying the angiotensin-stimulated calcium signal in bovine glomerulosa cells maintained in primary monolayer culture. As reported in previous studies employing acutely dispersed glomerulosa cells (Braley et al., 1984; Capponi et al., 1984; Kojima and

Ogata, 1986) angiotensin II produced a rapid increase in [Ca2+], in cultured cells (Fig. 1; Kramer, 1988). In contrast to that of acutely dispersed cells, however, the angiotensin-stimulated calcium signal in cultured cells was characterized by two distinct components, an initial, transient phase and a secondary, sustained plateau. The results presented here demonstrate directly that the initial component of the calcium signal produced by angiotensin II in cultured glomerulosa cells is derived from an intracellular calcium pool(s). The initial increase in [Ca2’], was manifested fully in the absence of extracellular calcium. Moreover, the increase in [Ca2’], produced by angiotensin II in the absence of extracellular calcium was inhibited by TMB-8 and dantrolene, agents presumed to block the mobilization of intracellular calcium stores (Putney and Bianchi, 1974; Chiou and Malagodi, 1975; Van Winkle, 1976; Clapper and Lee, 1985). Kojima and associates (Kojima and Ogata, 1986; Kojima et al., 1986) noted similar results in acutely dispersed bovine glomerulosa cells. Dantrolene has been shown to inhibit inositol trisphosphate (IP,-induced release of calcium in saponin-permeabilized cells (Kojima et al., 1984) and angiotensin II promoted the hydrolysis of phosphatidylinositides and concomitant accumulation of IP, in glomerulosa cells (Farese et al., 1984; Kojima et al., 1984; Enyedi et al., 1985). Together these observations indicate that an IP,-dependent release of calcium from an intracellular site, presumably the endoplasmic reticulum, is primarily responsible for the onset of the angiotensin-stimulated calcium signal. Such an action is consistent with that proposed for angiotensin II in a number of other cell types (Berridge, 1984; Smith et al., 1984; Thomas et al., 1984; Alexander et al., 1985). Closely associated with the onset of the calcium signal was an increase in calcium efflux. The present results extend previous observations (Elliott and Goodfriend, 1981; Williams et al., 1981; Foster and Rasmussen, 1983; Elliott et al., 1985; Kojima et al., 1985~) that angiotensin II promoted calcium efflux in acutely dispersed cells and provide the first direct demonstration that calcium efflux occurs concomitantly with an elevation in [Ca”],. In addition, angiotensin II caused an initial dose-dependent increase in the rate of

208

calcium efflux that was proportions to the release of intra~llular calcium. The kinetics of angiotensin-stimulated calcium efflux closely paralleled those of the intracellular calcium-dependent component of the calcium signal. For example, the intense increase in [Ca2’], produced by 10m9 M angiotensin II was accompanied by an increase in fractional calcium efflux from approximately 3.5% to 20%. Interestingly, the maximal calcium efflux achieved in response to lop9 M angiotensin II in cultured glomerulosa cells was 6- to 7-fold greater than that noted previously in dispersed cells, but comparable to the depletion in total intracellular calcium content produced by the peptide (Elliott et al., 1985; Kojima et al., 1985a). Although other homeostatic mechanisms may be involved, the marked increase in calcium efflux may account for the secondary decline in [Ca2’], characteristic of the calcium signal produced by high concentrations of angiotensin II. The finding that lanthanum blocked both calcium efflux and the decline in [Ca2’], supports this contention. As [Ca”], returned toward control levels there was a parallel reduction in the rate of calcium efflux. A concentration (lo-“r M) of angiotensin 11 that caused a less intense increase in intracellular calcium release also caused a proportionately smaller increase in calcium efflux. The calcium signal produced by lo- i1 M angiotensin II was also characterized by a more gradual elevation in [Ca”],. These observations suggest that the relative rates of intracellular calcium release and calcium efflux determine the specific ch~acte~stics of the initial phase of the an~otensin-stimulated calcium signal. The rate of calcium efflux also appears to contribute to the [Ca’+], achieved during the sustained phase of the calcium signal. Calcium efflux in cells treated with angiotensin II remained at a level 2.5- to 3-fold greater than that of control cells. These results differ from those of previous studies using acutely dispersed cells (Foster and Rasmussen, 1983; Rojima et al., 1985~): but a sustained increase in calcium efflux would be predicted given a sustained elevation in calcium influx (Kojima et al., 1985a, b) and a plateau in [Ca2’], (Kramer, 1988). Interestingly, the sustained increase in calcium efflux produced by angiotensin II at a concentration of lo-” M was

similar to that produced at 1O-9 M. This contrasts with initial changes in calcium efflux, but may reflect the fact that the magnitudes of the sustained calcium signals produced by the peptide at these concentrations were comparable. The mechanism underlying the sustained component of the angiotensin-stimulated calcium signal appears to be an influx of extracellular calcium. Support for such a mechanism is provided by the demonstration that the sustained elevation in [CaZ+], produced by angiotensin II is absent when cells are superfused with a calcium-free buffer, reversed by the removal of extraceflular calcium, and reinstated following repletion of extracellular calcium. The present results support those of Kojima et al. (1987) indicating that the angiotensin-sensitive intracellular calcium pool is limited in size and clearly demonstrate that intracellular calcium release alone can not sustain the elevation in [Ca2’],. In addition, nifedipine has been shown to attenuate the an~otensin-stimulated calcium signal in dispersed bovine (Capponi et al., 1984) and rat (I3raley et al., 1986) glomerulosa cell. The possibility that a sustained IP,-mediated mobilization of intracellular calcium is dependent upon extracellular calcium, however, cannot be excluded (Kojima et al., 1984). Nonetheless, the data presented here are consistent with previous studies demonstrating that angiotensin-stimulated aldosterone secretion was dependent upon extracellular calcium (Fakunding et al., 1979; Fakunding and Catt, 1980; Schiffrin et al., 1981). An~otensin II increased calcium influx in cultured glomerulosa cells, but such an effect was apparent only on the initial (O-10 s) rate of calcium uptake. Although other factors may contribute to the present results, the absence of a discernible effect of the peptide on calcium influx over a longer time interval may relate to the fact that the increase in calcium uptake occurred in the face of a concomitant increase in calcium efflux. Such a conclusion is supported by the observation that at times (O-2 min) when calcium efflux was maximal, an increase in calcium influx was not apparent. In cells exposed to angiotensin II for longer periods of time (5 or 15 min), in contrast, the initial rate of calcium uptake was 1.5- to 2-fold greater than that of control cells. Calcium efflux had significantly declined during the same time

intervals. The observation (Kramer, unpublished) that potassium at a concentration (20 mM) that produced a sustained elevation in [Ca2+]. comparable to that produced by angiotensin II (lo-” or lop9 M) - produced effects on initial calcium uptake similar to those of the peptide lends credence to the present results. Additional support is provided by reports of some (Kojima et al., 1985a, b), but not all (Elliott and Goodfriend, 1981; Elliott et al., 1985), investigators that angiotensin II increased calcium uptake in dispersed glomerulosa cells. At the concentrations examined in the present studies, no dose-dependent effect of angiotensin II on initial calcium uptake was observed. However, as suggested for the effects of the peptide on calcium efflux, the lack of a dose-response relationship may reflect the fact that these concentrations of angiotensin II produced similar increases in [Ca”], during the sustained phase of the calcium signal. Regardless, angiotensin II appears to produce a sustained increase in calcium influx that coincides with the sustained component of the calcium signal. Based on the results of these and other recent studies (Kramer, 1988) using bovine glomerulosa cells maintained in primary monolayer culture, it is proposed that angiotensin II produces multiple effects on calcium mobilization that result in dose-dependent and sustained elevations in [Ca2’],. The increase in [Ca2+], produced by angiotensin II is initiated by a rapid mobilization of calcium from an intracellular store and sustained by a prolonged effect of the peptide to promote the influx of extracellular calcium. An effect of angiotensin II to promote calcium efflux contributes to the magnitude and kinetics of the initial phase of the calcium signal as well as the final [Ca2’], achieved during the sustained phase. As proposed previously (Kojima et al., 1984, 1985a), the increase in [Ca2’], is presumed to initiate the actions of angiotensin II. In addition, the results presented here suggest that a sustained elevation in [Ca2+], is also important in maintaining the effects of angiotensin II on the glomerulosa cell. Studies are now required to define further the mechanisms by which angiotensin II affects calcium mobilization as well as the relationships between calcium and other angiotensin-generated second messengers.

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