(Received for publication, May 20, 1994; and in revised form, October 21, 1994)
From the
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.
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(12) or
ET
(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.
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
]
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
.
The data shown were reproduced in at least three independent
experiments.
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.
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]
, 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.
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 (
), 0.5
mM (
), 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
]
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
-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
]
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 (
) or 100 nM bradykinin (
) 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
]
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.
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.
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]
, we elicited a transient
rise in [Ca
]
by mechanisms
other than G protein activation. To provoke a Ca
transient in an inositol phosphate-independent manner,
thapsigargin was applied which increases
[Ca
]
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
]
is necessary for the subsequent decrease of total cell Ca.
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.
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]
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
]
. 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
]
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
]
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
]
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
]
(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
]
(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
]
(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
]
(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
]
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
]
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
]
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.
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
]
(31, 32) .
The rise in [Ca
]
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
]
(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
]
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]
. 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
]
evoked by the second
stimulus were diminished. A suppression of the transient rise in
[Ca
]
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-mediated response. This is
particularly important since the IP
receptor is reported to
be barely desensitized by common pathways, i.e. repeated
stimulation by IP
(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.