©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of Bradykinin and Endothelin-1 on the Calcium Homeostasis of Mammalian Cells (*)

(Received for publication, May 20, 1994; and in revised form, October 21, 1994)

Ursula Quitterer Christian Schröder Werner Müller-Esterl Hubert Rehm

From the Institute for Physiological Chemistry and Pathobiochemistry, Duesbergweg 6, D-55099 Mainz, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ca mobilization from intracellular stores is a major event in the signaling cascade triggered by peptide hormone receptors. The transient rise in intracellular free Ca concentration ([Ca]) is well characterized, but little is known about alterations of total cell Ca. Therefore we established a technique to determine changes in total cell Ca during hormone stimulation of Ca-loaded cells. Bradykinin and endothelin-1 reduced total cell Ca by up to 56% in HF-15 cells, COS-7 cells, and CHO K1 cells transfected with the rat B2 receptor cDNA. In Rat-1 cells and PC-12 cells, stimulation with endothelin-1 or bradykinin did not result in a net decrease in total cell Ca at physiological extracellular Ca concentration. Decrease in total cell Ca was preceded by an increase in [Ca] and blunting of the transient rise in [Ca] by a Ca chelator prevented the hormone-induced decrease in total cell Ca. Previous reduction of total cell Ca by one hormone suppressed the transient rise in [Ca] induced by another. The data present evidence that the hormones bradykinin and endothelin-1 are capable of switching off the Ca-mobilizing signal transduction pathway in a cell by depleting intracellular Ca stores. This process is accompanied by a significant reduction of total cell Ca.


INTRODUCTION

Bradykinin and endothelin-1 belong to families of vasoactive peptide hormones which are liberated by proteolytic cleavage of the precursor proteins kininogens (1, 2) and proendothelin(3) , respectively. Bradykinin, a vasodilator peptide(4) , affects epithelial ion transport(5) , induces inflammation processes(6) , and contracts smooth muscles(7) . Endothelin-1, a vasoconstrictor peptide(8) , regulates renal and pulmonary functions, and elevated levels of endothelin-1 are seen in a wide spectrum of pathological conditions (9) . The two peptide hormones act via G protein-coupled receptors, the bradykinin B2 receptor (10, 11) and endothelin ET(A)(12) or ET(B)(13) receptor, respectively. Binding of the hormones to their respective receptors induces a cascade of events, e.g. activation of phospholipase C, production of inositol trisphosphates, and release of Ca from intracellular stores(14, 15) . The resulting transient rise in [Ca] modulates mitogenesis and cell proliferation(16, 17) .

Several reports showed that bradykinin and endothelin-1 induce a Ca efflux from cells preloaded with Ca (18, 19) but total cell Ca was not determined. Because total cell Ca is linked to the cellular signaling cascades and to the regulation of cellular growth and differentiation(20) , we considered the analysis of hormone-induced changes in total cell Ca critical to an understanding of the cellular effects of bradykinin and endothelin-1. Here we report that bradykinin and endothelin-1 decrease up to 56% of total cell Ca. The decrease in total cell Ca renders the cell refractory to iterative Ca mobilization.


EXPERIMENTAL PROCEDURES

Materials

BAPTA/AM, (^1)fura-2/AM, ionomycin, pluronic F-127, and thapsigargin were from Calbiochem; (3-[I]iodotyrosyl)endothelin-1 (specific activity 2000 Ci/mmol), [2,3-prolyl-3,4-^3H]bradykinin (specific activity 98 Ci/mmol), and [Ca]Cl(2) (specific activity 10.58 mCi/mg) from Du Pont de Nemours; G-418 sulfate (Geneticin) from Life Technologies, Inc., Lipofectin was from Syntex, Aquasafe 300 from Packard; bradykinin, [desArg^9]bradykinin, endothelin-1, and BQ-123 from Saxon Biochemicals; bacitracin and enalapril from Sigma. Other chemicals were of analytical grade.

Cell Transfection

The 1.8-kilobase pair EcoRI fragment containing the entire coding region of the rat B2 receptor (10) was subcloned into the pcDNAI/Neo expression vector (Invitrogen). The resultant expression plasmid was stably transfected into CHO K1 cells by the Lipofectin transfection method(21) . Briefly, CHO K1 cells (1 times 10^6) were incubated with the Lipofectin-DNA complex (4 µg of DNA and 30 µl of Lipofectin) for 6 h at 37 °C. The reaction was terminated by the addition of 10% (v/v) fetal calf serum. Resistant clones were selected with G-418 (0.4 mg/ml) for 3-4 weeks. Subclone rB2CHO12/4 showed a maximum [^3H]bradykinin binding activity of 1.3 pmol/mg of protein at passage 2.

Cell Culture

Human foreskin fibroblasts (HF-15) expressing bradykinin B2 receptors (22) were grown for 2 to 3 weeks in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum. Cells were used at passages 9 to 15. CHO K1 (ATCC, CCL 61) and rB2CHO12/4 were grown in Ham's F12 medium containing 10% (v/v) fetal calf serum. Transfected cell line rB2CHO12/4 was used at passages 2 to 15. Rat-1 cells were grown in Dulbecco's modified Eagle's medium with 10% (v/v) fetal calf serum. PC-12 cells (ATCC, CRL 1721) were grown in Dulbecco's modified Eagle's medium including 10% (v/v) horse serum, 5% (v/v) fetal calf serum, and used at passages 5 to 15. COS-7 cells (ATCC, CRL 1651) were grown in Ham's F12 medium with 10% (v/v) fetal calf serum. All cell lines were kept at 37 °C in a humidified 5% CO(2),95% air atmosphere.

Binding of [^3H]Bradykinin

Cells were grown to confluency on 24-well plates. The medium was changed to 0.45 ml of minimum essential medium buffered with 20 mM Na-HEPES, pH 7.4 (HMEM) containing 2 mM bacitracin, 2 µM enalapril, and 0.05 mM Ca. For the binding assay, 50 µl of [2,3-prolyl-3,4-^3H]bradykinin in HMEM was added; final concentrations of [^3H]bradykinin ranged from 10 pM to 40 nM. For the determination of nonspecific binding, 10 µM (40 µM for COS-7 cells) unlabeled bradykinin was present. After 15 min (Rat-1 cells, PC-12 cells) or 30 min (CHO K1 cells, HF-15 cells, COS-7 cells) of incubation at 37 °C, the medium was suctioned, and the cells were washed three times with 1 ml of ice-cold incubation medium. Cells were dissolved in 0.5 ml of 0.5% (w/v) NaOH and analyzed in a beta-counter using 4 ml of the scintillation mixture (Aquasafe 300). Determinations were done in duplicate, and the results are the means of three separate experiments.

Binding of I-Endothelin-1

Cells were grown to confluency on 96-well plates. The medium was changed to 90 µl/well of HMEM containing 0.05 mM Ca, and 10 µl of (3-[I]iodotyrosyl)endothelin-1 in HMEM was added to final concentrations ranging from 10 pM to 5 nM. Nonspecific binding was determined in the presence of 5 µM unlabeled endothelin-1. After 2 h of incubation at 37 °C, the medium was suctioned and the cells were washed three times with 0.3 ml of ice-cold incubation medium. Cells were dissolved in 0.1 ml of 0.5% (w/v) NaOH, and the radioactivity was analyzed in a -counter. Determinations were done in duplicate, and the results are the means of three separate experiments.

Determination of [Ca](i) by Fura-2 Assay

Cells on 10-mm diameter glass coverslips were grown to confluency. They were washed twice with buffer A (minimum essential medium buffered with 20 mM Na-HEPES, pH 7.4, without vitamins; alpha-D-glucose was added before use; for quantitative Ca measurements, phosphate was omitted; the Ca concentration is indicated). For fura-2 loading, cells were incubated in buffer A containing 2 µM fura-2/AM and 0.04% (w/v) of the nonionic detergent pluronic F-127 for 30 min at 30 °C. They were washed twice with buffer A and stored in buffer A at room temperature for another 30 min to allow for complete de-esterification of fura-2/AM. Except for handling, cells were kept in the dark.

During the measurement, the coverslips were fixed at an angle of 45° in a holder and placed into a 37 °C thermostatted quartz cuvette. Fluorescence was measured with a Hitachi F4500 fluorescence photometer. The excitation wavelength alternated in intervals of 600 ms between 340 and 380 nm. The slit width was 10 nm, and the emission was measured at 510 nm. The intracellular free Ca concentration [Ca](i) was calculated from the ratio of 340/380 nm as described(23) . To determine the fluorescence of Ca-free fura-2 in the cells, we loaded the cells with fura-2 and a 5-fold excess of BAPTA. For calibration, the cells were exposed to 2 to 10 µM ionomycin in the presence of 1.8 mM extracellular Ca (final concentrations). The background fluorescence was determined by adding 5 mM MnCl(2). The data shown were reproduced in at least three independent experiments.

Determination of Ca Uptake

Cells were grown to confluency on 24-well plates and washed twice with HMEM. To each well, 0.5 ml of HMEM containing Ca as indicated was added, and the cells were kept for 2 h at 37 °C in a humidified atmosphere without CO(2). The Ca uptake was started by the addition of 50 µl of Ca (2.5 µCi) in HMEM. The medium was mixed by pipetting three times up and down a 250-µl pipette. After different times, the medium was removed by suction, and the cells were washed three times with 1 ml of ice-cold La buffer (15 mM Na-HEPES, pH 7.4, 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl(2), 1.8 mM CaCl(2), 0.2 mM LaCl(3), 0.01% (w/v) bovine serum albumin). The washed cells were dissolved in 0.5 ml of 0.5% (w/v) NaOH and counted. Determinations were done in duplicate, and the results are the means of three independent experiments.

Determination of Total Cell Ca

Cells were seeded into 24-well plates and grown to confluency. They were washed twice with Ca-free HMEM, and 0.44 ml of HMEM containing Ca as indicated was added to each well. For parallel measurements of [Ca](i) and total cell Ca, HMEM was replaced by buffer A throughout the experiment. The cells were kept for 2 h at 37 °C in a humidified atmosphere without CO(2). Each well then received 10 µl of Ca (1 µCi; 4 µCi for experiments with extracellular Ca at or higher than 0.5 mM). The medium was mixed by pipetting it three times up and down a 250-µl pipette, and the cells were incubated for 4 h (unless otherwise indicated) at 37 °C. Then, 50 µl of hormone in HMEM or medium alone (control) was added, mixed as before, and incubated. The medium was suctioned after the time indicated, and the cells were washed three times with 1 ml of ice-cold La buffer. The cells were dissolved in 0.5 ml of 0.5% (w/v) NaOH and counted in a beta-counter (Packard) with 4 ml of scintillator. Determinations were done in duplicate. Because Ca in the cell was in equilibrium with the extracellular medium, we regarded the Ca of the cells as a marker of total cell Ca.

Simultaneous Measurement of Ca Extrusion and of [Ca](i)

Cells were grown to confluency on 6-well plates containing 10-mm diameter glass coverslips. The medium was suctioned, and the cells were washed twice with Ca-free buffer A (see ``fura-2 assay''). Two ml of buffer A was added; the Ca concentration was 0.05 mM throughout the experiment. The cells were kept for 1 h at 37 °C in a humidified atmosphere without CO(2). To each well, 40 µl of Ca (16 µCi) was then added, and the cells were kept at 37 °C for 2h. Buffer A was suctioned and 2 ml of fresh buffer A containing 2 µM fura-2/AM and 0.04% (w/v) pluronic F-127 was added. Each well received another 40 µl of Ca (see above), followed by an incubation for 45 min at 30 °C. The coverslips were washed three times with buffer A, immediately mounted in a holder, and placed into a 37 °C thermostatted quartz cuvette containing 2 ml of buffer A. Fluorescence was determined as above. At the time points indicated, hormone or buffer alone (control) was added, and 100-µl aliquots were removed from the cuvette and counted; the volume of the cuvette was readjusted by addition of fresh buffer A. Basal leakage of Ca from the cells was determined on cells stimulated with buffer alone and subtracted from each value. The presented data were obtained in at least four independent experiments.

Determination of Protein Concentration

Protein in the NaOH extracts was determined by the BCA assay (Pierce) using bovine serum albumin as the standard. NaOH had no influence on the color development.


RESULTS

Transient Rise in [Ca](i)

To analyze the effect of bradykinin and endothelin-1 on Ca homeostasis, we chose two cell lines: human foreskin fibroblasts (HF-15) and rat fibroblasts (Rat-1). Each cell line expressed both hormone receptors (cf. Table 3). A transient rise in [Ca](i) was elicited by bradykinin (Fig. 1, A and C) and endothelin-1 (Fig. 1, B and D) at an extracellular Ca concentration of 1.8 mM. The signals differed in the peak values, the height of the plateau following the peak and the time to reach this plateau. The highest peak was obtained with bradykinin and HF-15 cells, and the lowest peak with bradykinin and Rat-1 cells (Fig. 1, A and C). The time of decline from the peak to plateau level was 60 to 80 s for bradykinin and endothelin-1 in HF-15 cells and for bradykinin in Rat-1 cells (Fig. 1, A, B, and C). For endothelin-1 and Rat-1 cells, the time to reach plateau was longer than 4 min (Fig. 1D). This long decline phase is particularly susceptible to a decrease in the extracellular Ca concentration (own observation, cf. Fig. 7and Fig. 8and (24) ), and thus endothelin-1 may induce a major Ca influx from the extracellular space.




Figure 1: Bradykinin- and endothelin-induced transient rise in [Ca] in HF-15 and in Rat-1 cells. HF-15 (A and B) and Rat-1 cells (C and D) on glass coverslips were loaded with fura-2, and [Ca] was determined. The extracellular Ca concentration was 1.8 mM. At the time point indicated, 10 nM bradykinin (A), 10 nM endothelin-1 (B and D), or 100 nM bradykinin (C) was added to the cells. For details, cf. ``Experimental Procedures.''




Figure 7: Sequential hormone stimulation. HF-15 cells (A and B) or Rat-1 cells (C and D) grown on glass coverslips were loaded with fura-2, and [Ca] was determined. The extracellular Ca concentration was 1.8 mM. At the time points indicated, hormone was added to a final concentration of 10 nM. The bradykinin concentration with Rat-1 cells was 100 nM (C and D). In a parallel experiment (extracellular Ca concentration was 1.8 mM), total cell Ca was determined on Ca-equilibrated cells at the time points indicated. Error bars indicate S.D. of a single experiment.




Figure 8: Sequential hormone stimulation at 0.05 mM extracellular Ca concentration of Rat-1 cells. The same experimental setting as in Fig. 7was applied except that the extracellular Ca concentration was 0.05 mM.



Determination of Total Cell Ca

Does total cell Ca change during the hormone-induced transient rise in [Ca](i)? To determine total cell Ca, we loaded cells with Ca to equilibrium. Equilibration times (t) for HF-15 cells were 45, 15, and 15 min at extracellular Ca concentrations of 0.05, 0.5, and 5 mM, respectively. Equilibration times of other cell lines tested did not differ significantly from HF-15 cells (not shown). Once equilibrium was reached (leq3 h), the Ca content of the cells did not change over an observation period of 5 h (not shown).

Addition of 10 nM bradykinin to HF-15 cells (in equilibrium with Ca) resulted in a loss of up to 44% (6.1 ± 1 nmol/mg of protein) of total cell Ca within 4 min (Fig. 2A). The initial value of total cell Ca was 13.9 ± 1.5 nmol/mg of protein which is in agreement with data obtained for, e.g. nervous tissue(25) . Stimulation with endothelin-1 decreased total cell Ca, although to a lesser extent, i.e. only by 32% (4.4 ± 0.5 nmol/mg of protein). After a decrease in total cell Ca, the cells refilled with Ca. In the presence of endothelin-1, refilling was complete after 40 min, whereas in the presence of bradykinin after 1 h only 76% of the control value was reached. Thus in HF-15 cells, bradykinin and endothelin-1 produce a profound and enduring decrease in total cell Ca.


Figure 2: Measurement of total cell Ca in HF-15 (A and B) and in Rat-1 (C and D) cells. Cells were loaded with Ca to equilibrium. The extracellular Ca concentration was 1.8 mM. At t = 0, hormone or buffer as a control was added. At the time points indicated, total cell Ca was determined in the presence of 10 nM bradykinin (A), 10 nM endothelin-1 (B and D), or 100 nM bradykinin (C). Control values for each time point are given (). Each point is the mean (± S.D.) of three separate experiments.



In Rat-1 cells, bradykinin did not influence total cell Ca (Fig. 2C). Endothelin-1 induced a small but significant increase in total cell Ca of 1.7 ± 0.2 nmol/mg of protein within 4 min (Fig. 2D).

Although in both cell lines bradykinin and endothelin-1 induced a transient rise in [Ca](i), their total cell Ca responded differently to hormone stimulation: in HF-15 cells bradykinin and endothelin-1 reduced total cell Ca, whereas in Rat-1 cells bradykinin did not influence total cell Ca, and endothelin-1 transiently increased total cell Ca.

Influence of Extracellular Ca on the Hormone-induced Decrease in Total Cell Ca

What processes are responsible for the changes in total cell Ca? Two components determine the transient rise in [Ca](i): the release of Ca from intracellular stores and the influx of Ca from the extracellular medium. To determine the contribution of the Ca influx to the changes in total cell Ca, we measured the transient rise in [Ca](i) and the decrease in total cell Ca at different extracellular Ca concentrations (Fig. 3, A and B). In HF-15 cells, the peak value for the bradykinin-induced transient rise in [Ca](i) did not change with the extracellular Ca concentration but the plateau value increased from 48 to 170 nM when the extracellular Ca concentration was raised from 0.05 to 5 mM. This indicates an increasing Ca influx with increasing extracellular Ca concentration (Fig. 3A). Further, the time to reach the plateau was significantly longer at higher extracellular Ca, i.e. 50 s, 80 s, and 120 s at 0.05, 0.5, and 5 mM extracellular Ca, respectively.


Figure 3: Influence of extracellular Ca concentration on the transient rise in [Ca] and on the decrease in total cell Ca. HF-15 cells were loaded with fura-2, and [Ca] was determined. The extracellular Ca concentration was 5, 0.5, or 0.05 mM. At t = 40 s, bradykinin (100 nM) was added and [Ca] was determined (A). For measurement of total cell Ca, cells were loaded with Ca. The extracellular Ca concentration was 5 mM (bullet), 0.5 mM (circle), or 0.05 mM (X). At t = 0, bradykinin (100 nM) was added, and total cell Ca was determined at the time points indicated (B). For control values, buffer alone was added. The controls did not significantly differ from the value obtained for t = 0 (not shown). Each point is the mean (± S.D.) of three separate experiments.



In a parallel experiment, total cell Ca was determined. In the absence of hormone, total cell Ca increased with the extracellular Ca concentration, the values ranging from 3.8 ± 0.2, 9 ± 0.3, to 19.5 ± 1.2 nmol/mg of protein at 0.05, 0.5, and 5 mM extracellular Ca, respectively. Since [Ca](i) does not change with extracellular Ca (Fig. 3A), the increase in total cell Ca must be due to an increase of Ca in the intracellular stores (i.e. mitochondria and endoplasmic reticulum/IP(3)-sensitive compartment). In the presence of 100 nM bradykinin, total cell Ca decreased even at an extracellular Ca concentration of 5 mM. The actual decrease was 2 ± 0.1, 4.4 ± 0.2, and 6.4 ± 0.6 nmol/mg of protein at 0.05, 0.5, and 5 mM extracellular Ca, respectively.

At extracellular Ca concentrations of 0.05 mM, 0.5 mM, or 5 mM total cell Ca reached minimum values after 1, 4, and 10 min of bradykinin challenge, respectively (Fig. 3B). Thereafter, the cells refilled with Ca, and this process was completed after 30 min at 5 mM extracellular Ca. At 0.5 or 0.05 mM extracellular Ca, only 75% or 48%, respectively, of the initial total cell Ca was reached after 1 h (Fig. 3). Full restoration of total cell Ca was seen after 2 h of incubation (not shown). The different kinetics at different extracellular Ca concentrations indicates that, in HF-15 cells, Ca extrusion prevails over hormone-activated Ca influx. This results in a net decrease of total cell Ca even at an extracellular Ca concentration of 5 mM.

What happens in Rat-1 cells when Ca influx is suppressed by low extracellular Ca? To avoid Ca leakage out of the cell via activated Ca channels, we chose an extracellular Ca concentration of 0.05 mM, which is sufficiently higher than the [Ca](i) of about 50-100 nM. Stimulation with 10 nM endothelin-1 at 0.05 mM extracellular Ca decreased total cell Ca from 2.7 ± 0.3 nmol/mg of protein to 0.9 ± 0.1 nmol/mg of protein (Fig. 4). This is in contrast to physiological extracellular Ca, where no reduction of total cell Ca was seen (cf. Fig. 2D). Thus, in Rat-1 cells at low (0.05 mM) extracellular Ca, the process of Ca extrusion prevails over Ca influx, whereas at physiological (1.8 mM) extracellular Ca the process of Ca influx prevails over Ca extrusion leading to a net increase in total cell Ca (cf. Fig. 2D).


Figure 4: Total cell Ca of Rat-1 cells at 0.05 mM extracellular Ca concentration. Rat-1 cells were loaded with Ca to equilibrium, and 10 nM endothelin-1 (bullet) or 100 nM bradykinin (circle) was added at t = 0. At the time points indicated, total cell Ca was determined. For control, buffer alone was added. Control values did not significantly differ from the values obtained in the presence of 100 nM bradykinin (not shown). Each point is the mean (± S.D.) of three separate experiments.



Bradykinin (100 nM) did not alter total cell Ca of Rat-1 cells, even at 0.05 mM extracellular Ca. The elevation of [Ca](i) to 160 nM by bradykinin (cf. Table 3) during the cytoplasmic Ca transient seems to be below the threshold of the Ca pumps which extrude Ca out of the cell.

Simultaneous Measurement of Ca Extrusion and of the Transient Rise in [Ca](i) in Rat-1 Cells

To verify that Ca does not leave the Rat-1 cells during the bradykinin-elicited transient rise in [Ca](i), we simultaneously measured the transient rise in [Ca](i) and Ca extrusion from the cells into medium that contains only Ca (0.05 mM). In this setting, bradykinin induced a transient rise in [Ca](i) in Rat-1 cells but no significant Ca extrusion (Fig. 5A) indicating that in Rat-1 cells the Ca pumps are not active at a cytoplasmic Ca concentration of up to 160 nM. The Ca extrusion mechanisms in these cells were intact because stimulation of Rat-1 cells with endothelin-1 induced a transient rise in [Ca](i) and, concomitantly, Ca extrusion (Fig. 5B).


Figure 5: Simultaneous measurement of Ca extrusion and of the transient rise in [Ca]. Rat-1 cells were grown on glass coverslips and loaded with Ca and fura-2 (concentration of Ca was 0.05 mM). At the time points indicated, 100 nM bradykinin (A) or 10 nM endothelin-1 (B) was added, and [Ca] was determined; simultaneously, the Ca content of the medium was monitored. The basal leakage of Ca from the cells was determined in the presence of buffer alone and subtracted from each value.



The Role of the Ca Transient for the Decrease in Total Cell Ca

The decisive process for the decrease in total cell Ca is the extrusion of Ca from the cell. The experiment with bradykinin in Rat-1 cells showed that the peak value of the transient rise in [Ca](i) may be critical for the activation of the plasma membrane Ca pumps (i.e. Ca-ATPases, Na/Ca exchanger) (cf. Fig. 5A). Therefore, we tried to clarify if the transient rise in [Ca](i) is necessary for the hormone-induced decrease in total cell Ca. In HF-15 cells, the transient rise in [Ca](i) was prevented by the nonfluorescent Ca chelator BAPTA, and, simultaneously, Ca extrusion was measured (Fig. 6A). When the cells were incubated with 10 µM BAPTA/AM for 15 min, bradykinin (10 nM) did not elicit a transient rise in [Ca](i), whereas without BAPTA/AM, bradykinin induced Ca extrusion in parallel to the transient rise in [Ca](i) (Fig. 6B). Blocking of the transient rise in [Ca](i) also blocked Ca extrusion.


Figure 6: Simultaneous measurement of Ca extrusion and of the transient rise in [Ca] in the presence of BAPTA. HF-15 cells were loaded with fura-2 and Ca. At the time point indicated, 10 nM bradykinin was added. In A, the cells were preincubated with 10 µM BAPTA/AM for 15 min. For determination of [Ca] and of Ca extrusion, the experimental setting of Fig. 5was applied.



The measurement of total cell Ca in the presence of intracellular BAPTA confirmed this result. In the presence of BAPTA, total cell Ca did not decrease (Table 1). This experiment was, similarly to Fig. 6, performed at an extracellular Ca concentration of 0.05 mM.



To further analyze the relationship between decrease in total cell Ca and the transient rise in [Ca](i), we elicited a transient rise in [Ca](i) by mechanisms other than G protein activation. To provoke a Ca transient in an inositol phosphate-independent manner, thapsigargin was applied which increases [Ca](i) by the inhibition of endoplasmic reticulum Ca-ATPases(26) . Thapsigargin decreased total cell Ca in HF-15 cells (51% of the control value), although to a lower extent than bradykinin (Table 1). The effects of thapsigargin and bradykinin were not additive. We conclude that the transient rise in [Ca](i) is necessary for the subsequent decrease of total cell Ca.

Specificity of Hormone-induced Decrease of Total Cell Ca

It remains to be demonstrated that the hormoneinduced decrease in total cell Ca is specific for bradykinin or endothelin receptors. In HF-15 cells, HOE140, a potent antagonist of the B2 receptor(27) , inhibited the decrease in total cell Ca induced by bradykinin but had no effect per se (Table 2). A B1 bradykinin receptor agonist, [desArg^9]bradykinin, did not act on total cell Ca nor did it interfere with the bradykinin-mediated Ca decrease. We conclude that bradykinin decreases total cell Ca of HF-15 cells via the bradykinin B2 receptor. The specificity of endothelin-induced Ca decrease was established by the endothelin ET(A) receptor antagonist BQ-123 (28) which completely blocked the endothelin-mediated decrease in total cell Ca of HF-15 cells (Table 2).



Bradykinin- and Endothelin-induced Changes in Total Cell Ca of Several Cell Lines

Additional cell lines were tested to find out if the hormone-induced decrease in total cell Ca is a general phenomenon. We chose COS-7 cells derived from kidney and rat pheochromocytoma cells (PC-12) which are related to sympathetic neurons. Both cell lines endogenously express bradykinin receptors. In addition, CHO K1 cells were transfected with rat B2 receptor cDNA (rB2CHO12/4). The binding parameters are summarized in Table 3. In all cell lines, bradykinin induced a transient rise in [Ca](i). Among the cell lines tested, Rat-1 cells and HF-15 cells were the only ones which responded to endothelin-1, also (see above). The cell lines differed in their responsiveness and their maximum signals. Also, at low extracellular Ca (0.05 mM), total cell Ca decreased upon bradykinin or endothelin-1 stimulation (except for bradykinin and Rat-1 cells, cf. Fig. 4). Between the dose-response curves for the hormone-induced transient rise in [Ca](i) and the decrease in total cell Ca, minor differences were seen. Whether these differences reflect differential activation of the two processes or whether they are due to the presence of fura-2 during the measurement of [Ca](i) needs further investigation.

At physiological extracellular Ca concentration (1.8 mM), bradykinin induced in rB2CHO12/4 cells a dramatic decrease in total cell Ca of 6.1 ± 0.5 nmol/mg of protein. In addition, bradykinin decreased total cell Ca in COS-7 cells, although to a lesser extent (2.5 ± 0.3 nmol/mg of protein). In PC-12 cells and in Rat-1 cells, total cell Ca did not change upon bradykinin stimulation.

As already shown (Fig. 2), endothelin-1 induced a decrease in total cell Ca in HF-15 cells (4.4 ± 0.5 nmol/mg of protein). In contrast, in Rat-1 cells, endothelin-1 provoked a net increase of total cell Ca (1.7 ± 0.2 nmol/mg of protein). In agreement with the lack of endothelin-1 binding sites in COS-7, nontransfected CHO K1 cells, and PC-12 cells, no change in total cell Ca was seen with this hormone.

Thus, the effect of bradykinin and endothelin-1 on total cell Ca is varying between mammalian cell lines.

Relationship of Bradykinin- and Endothelin-sensitive Ca Stores

The hormonal regulation of total cell Ca depends on the cell line. This raises the possibility that the decrease in total cell Ca may constitute a signal of its own. By a decrease of total cell Ca, the hormones decrease the Ca content of intracellular stores. Thus, the signal may regulate the answer of the cell to subsequent hormone stimuli.

After a decrease of total cell Ca by one hormone, we tried to stimulate the cells a second time by a second stimulus. In two cell lines (HF-15, Rat-1), both bradykinin and endothelin-1 induced a transient rise in [Ca](i) but different responses with respect to total cell Ca (cf. Fig. 1and Fig. 2). We applied bradykinin and endothelin-1 in a sequential order and measured total cell Ca and [Ca](i). The extracellular Ca concentration in this set of experiments was 1.8 mM. In a first experiment, 10 nM bradykinin evoked a maximum transient rise in [Ca](i) in HF-15 cells (Fig. 7A), thereby reducing total cell Ca by 6 nmol/mg of protein. When 10 nM endothelin-1 was added 4 min after bradykinin, the cells were unresponsive (Fig. 7A). In a second experiment, 10 nM endothelin-1 evoked a transient rise in [Ca](i) in HF-15 cells (Fig. 7B) and decreased total cell Ca by 3.5 nmol/mg of protein. When 10 nM bradykinin was added 4 min later, the transient rise in [Ca](i) was reduced (Fig. 7B), and the total cell Ca decreased only by an additional increment of 2.5 nmol/mg of protein. These results indicate that the decrease in total cell Ca induced by one hormone suppresses the Ca mobilization by a second hormone.

What happens with Rat-1 cells? Total cell Ca does not decrease in Rat-1 cells during hormone stimulation at 1.8 mM extracellular Ca (cf. Fig. 2). Application of 100 nM bradykinin resulted in a small, although significant, transient rise in [Ca](i) (Fig. 7C) but not in a decrease of the total cell Ca. When 10 nM endothelin-1 was added 4 min later, the maximum transient rise in [Ca](i) (Fig. 7C) and no decrease of the total cell Ca were observed. Then we changed the sequence of hormone stimulation. We first added 10 nM endothelin-1 which elicited a maximum transient rise in [Ca](i) (Fig. 7D) but no decrease in the total cell Ca. The subsequent addition of 100 nM bradykinin did evoke a significant Ca transient (Fig. 7D). Thus, there was no mutual interference of the Ca transients and no bradykinin- or endothelin-induced decrease of the total cell Ca.

To prevent refilling of intracellular stores we repeated the last experiment at 0.05 mM extracellular Ca. Endothelin-1 (10 nM) decreased total cell Ca from 2.7 to 0.9 nmol/mg of protein (Fig. 8A). If 100 nM bradykinin was applied 4 min later, it did not evoke a transient rise in [Ca](i) (Fig. 8A). Suppression of Ca influx revealed that bradykinin and endothelin-1 address overlapping stores.

When the sequence of hormones was changed, bradykinin evoked a significant Ca transient and no change in total cell Ca (Fig. 8B), and the endothelin-1 signal 4 min later was similar to Fig. 8A. Thus, it is obviously the decrease in total cell Ca that predicts that a further transient rise in [Ca](i) will be blunted.

To further analyze the mutual interference of bradykinin- and endothelin-1-induced Ca signals, we depleted Ca stores of HF-15 cells in a hormone receptor-independent manner, i.e. with thapsigargin. First the time course of thapsigargin-induced decrease in total cell Ca was established (extracellular Ca was 1.8 mM); total cell Ca was reduced by 4% after 1 min and by 50% after 4 min of exposure to thapsigargin (Fig. 9, B and D). In a set of two parallel experiments, bradykinin (Fig. 9, A and B) or endothelin-1 (Fig. 9, C and D) were added 1 min (Fig. 9, A and C) or 4 min (Fig. 9, B and D) after thapsigargin application. Then the respective transient rise in [Ca](i) was measured. Thapsigargin-induced reduction of the total cell Ca had a profound effect on the hormone-induced transients: decreasing the total cell Ca by thapsigargin significantly reduced the peak heights of the Ca transients evoked by bradykinin and endothelin-1 (Fig. 9, B and D). In contrast, stimulation with bradykinin or endothelin-1 at t = 120 s (1 min of thapsigargin stimulation) did not significantly reduce the transient rise in [Ca](i) of the two hormones. We conclude that previous depletion of intracellular Ca stores by one hormone renders the cell refractory to the stimulus of a second hormone.


Figure 9: Thapsigargin-induced decrease in total cell Ca and the transient rise in [Ca]. HF-15 cells were grown on glass coverslips and loaded with fura-2, and [Ca] was determined. The extracellular Ca concentration was 1.8 mM. At t = 60 s, thapsigargin (300 nM) was added. Time of hormone addition (10 nM) is indicated. In a parallel experiment, total cell Ca was monitored on Ca-loaded cells (extracellular Ca concentration was 1.8 mM). At the time points indicated, total cell Ca was determined. The dotted lines mark the total cell Ca at the time of hormone application. Error bars indicate S.D. of a single experiment.




DISCUSSION

Mobilization of intracellular Ca is an important event in the second messenger cascade of G protein-coupled receptors. It is well established that bradykinin and endothelin-1 release Ca from intracellular stores(29, 30) . In addition, a hormone receptor-mediated influx of Ca from the extracellular space contributes to the transient rise in [Ca](i)(31, 32) . The rise in [Ca](i) might account for the activation of membrane-bound Ca pumps which then mediate Ca efflux from the cells(33) . Indeed, several groups reported a bradykinin- or endothelin-induced Ca efflux from the cell(34, 35) . It was unknown whether the hormone-induced Ca efflux results in a change of the net Ca content of the cell or whether efflux is balanced by Ca entry(36) . Therefore, we established a method to quantitate net changes in the total cell Ca during hormone stimulation.

Our data obtained with various cell lines demonstrate that bradykinin and endothelin-1 decrease total cell Ca by up to 56%. So, the hormone-sensitive stores of these cell lines contain about half of the total cell Ca, and this amount can be extruded within 4 min. At physiological extracellular Ca, it takes more than 1 h to refill the depleted Ca stores. The mechanisms underlying this dramatic effect are not yet fully understood. But the transient rise in [Ca](i) (after exceeding a threshold) seems responsible for activating Ca extrusion: blunting of the transient abolished Ca extrusion and therefore abolished the decrease in total cell Ca. On the other hand, Ca influx counteracts Ca extrusion. Thus, total cell Ca decrease seems to be regulated by the transient rise in [Ca](i) and by Ca influx.

Is hormone-induced decrease in total cell Ca physiologically relevant? In HF-15 cells, bradykinin, endothelin-1, and thapsigargin induced both, a decrease in total cell Ca and a transient rise in [Ca](i). Application of one of these stimuli made the cell refractory to a second stimulus: the decrease in total cell Ca and the transient rise in [Ca](i) evoked by the second stimulus were diminished. A suppression of the transient rise in [Ca](i) by a second stimulus is in agreement with previous observations(37) . It was hypothesized to be due to a depletion of overlapping Ca stores by the first agent. Our experiments demonstrate for the first time that total cell Ca is indeed reduced after stimulation with thapsigargin, endothelin-1, or bradykinin. Interestingly, if total cell Ca is not reduced by the first hormone, the Ca transient of the second hormone remains unscathed: with Rat-1 cells and at physiological external Ca, total cell Ca remained constant or even increased during hormone challenge presumably due to massive influx of external Ca, and, therefore, the Ca transient of the second hormone was not altered. Nonetheless, even in Rat-1 cells, the stores of bradykinin are a subset of the endothelin-addressed stores. This overlapping of stores was revealed by the inhibition of influx at low external Ca (Fig. 8, A and B). Therefore, we conclude that it is the balance between Ca extrusion and Ca influx which determines the responsiveness to subsequent Ca-mobilizing signals. Measurement of total cell Ca indicates to which side the balance is tipped.

The hormone-regulated adaptation to heterogeneous stimuli shown by our experiments is of special interest as it provides a mechanism to ``desensitize'' the IP(3)-mediated response. This is particularly important since the IP(3) receptor is reported to be barely desensitized by common pathways, i.e. repeated stimulation by IP(3)(38) . This involvement of Ca in signal desensitization processes is analogous to the involvement of Ca in light adaptation(39) . The signal transduction pathway of the light receptor rhodopsin which belongs to the same superfamily of G protein-coupled receptors is modulated by Ca efflux enabling adaptation to a wide range of different light signals(40) . To fully understand the physiological impact of hormone-induced decrease in total cell Ca, further studies are needed focusing on the potential role of the decrease in total cell Ca on the Ca-triggered signal transduction pathway.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: BAPTA/AM, bis-(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid, tetra(acetoxymethyl) ester; [Ca], intracellular free Ca concentration; [Ca], extracellular Ca concentration; fura-2/AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2`-amino-5`-methylphenoxy)-ethane-N,N,N`,N`tetraacetic acid, pentaacetoxymethyl ester; HMEM, minimum essential medium buffered with 20 mM Na-HEPES, pH 7.4, containing Ca as indicated; HOE140, D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-Tic-Oic-Arg; Hyp, hydroxyproline; Thi, beta-2-thienylalanine; Tic, D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Oic, (3alphaS,7alphaS)-octahydroindol-2-carboxylic acid; BQ-123, cyclo(-D-Asp-Pro-D-Val-Leu-D-Trp-); IP(3), inositol 1,4,5-trisphosphate.


ACKNOWLEDGEMENTS

We thank Drs. A. A. Roscher (Munich) for HF-15 cells, K. Jarnagin (Palo Alto) for rat B2 receptor cDNA, M. Schmidt (Ludwigshafen) for Rat-1 cells, B. Schölkens (Frankfurt) for HOE140, and G. Reiser (Tübingen) for helpful discussions.


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