(Received for publication, August 1, 1994; and in revised form, November 30, 1994)
From the
Tissue factor (TF) is the most potent trigger of blood clotting
known. It activates factor VII (FVII) thereby initiating a cascade of
proteolytic reactions resulting in thrombin production. The cloning of
TF revealed its structural characteristics to be those of a receptor
related to the class 2 cytokine receptor superfamily, but until now no
intracellular signal has been discovered related to binding of the
ligand (FVIIa) to the putative receptor. We have studied possible
intracellular signaling effects of the FVIIa-TF interaction by
measuring cytosolic free Ca in single fura-2-loaded
cells and found that 200 nM FVIIa caused Ca
transients in about 30% of human umbilical vein endothelial cells
treated with interleukin-1
to express TF, compared to below 5% in
uninduced cells. A gradual increase of the basal Ca
level was also caused by binding of FVIIa. In the human bladder
carcinoma cell line J82, which has a high constitutive TF activity,
similar results were found. An antibody neutralizing TF activity
decreased the response rate to control levels. COS-1 cells which do not
make TF did not respond to FVIIa as opposed to COS-1 cells expressing
TF after transfection with a human TF cDNA construct. The canine kidney
cell line MDCK, a constitutive TF producer, responded especially well;
up to 100% of the cells examined showed Ca
oscillations which were dose dependent with regard to frequency,
latency, maximal amplitude, and recruitment of responding cells. The
frequency was reduced by inhibition of Ca
influx with
100 µM LaCl
. In confluent MDCK cells the
Ca
oscillations were synchronous, constituting the
first evidence of a synchronous cytosolic Ca
oscillator generated by global application of agonist. Thus, TF
mediates a cytosolic Ca
signal upon interaction with
its ligand FVIIa, thereby suggesting a more complex biological role for
TF.
Tissue factor (TF) ()is the most potent trigger of
blood clotting known(1, 2) . TF binds the plasma
coagulation factor VII (FVII), a vitamin K-dependent
glycoprotein(3) , leading to its proteolytic cleavage and
activation (FVIIa). This complex, in the presence of free
Ca
and phospholipid, will then catalyze the
activation of factor X or IX (for reviews see (4, 5, 6) ). The cloning of TF revealed its
structural characteristics to be those of a receptor, with a
219-amino-acid extracellular domain, a 23-amino-acid transmembrane
part, and a cytoplasmic tail of 21 amino
acids(7, 8, 9, 10) . Bazan (11) suggested that TF was structurally related to the class 2
cytokine receptor superfamily which includes the interferon (IFN)
/
and
receptors. The receptor concept requires this
structural aspect and in addition the existence of a high affinity-high
specificity ligand, which when binding to the receptor should result in
an intracellular signal. The functional ligand for TF is probably
FVII/FVIIa. The structural relationship to known receptors may give a
clue to possible signaling effects of the FVIIa-TF interaction. One
early signaling event in the IFN-
-mediated signal is an increase
of the cytosolic free Ca
concentration
([Ca
]
)(12, 13) .
The Ca
signal is brought about by both release from
internal stores and influx across the plasma membrane. Consequently, we
chose to study the effect of FVIIa binding to TF on
[Ca
]
in single cells
loaded with the fluorescent Ca
indicator fura-2 in a
digital imaging system. To look for such a Ca
response, we took advantage of the ability of interleukin-1
(IL-1
) to induce TF in HUVEC(14) . This allows the use of
uninduced HUVEC as controls. TF is expressed at maximum levels after 6
h of treatment with IL-1
(15) .
We report here the
detection of an increase in the intracellular level of Ca on binding of FVIIa to HUVEC induced by IL-1
to synthesize
TF. A similar increase in Ca
levels was detected upon
binding of FVIIa to TF in the human bladder carcinoma cell line J82
which produces TF constitutively and in monkey kidney fibroblasts
(COS-1) stably transfected with a human TF construct. J82 cells treated
with an antibody to neutralize TF activity and untransfected COS-1
cells which do not express TF showed no Ca
signal
upon exposure to FVIIa. The canine kidney cell line MDCK, a
constitutive TF producer, responded especially well to FVIIa.
TF activity was measured by a one-stage procoagulant assay (16) using pooled normal human plasma as substrate and by a
twostage assay (17) based on the activation of FX by the
TFFVIIa
Ca
membrane complex and
expressed as dOD (0-60 min) at 405 nm from the cleavage of the
chromogenic substrate. The results were compared to a standard curve
established by serial dilution of a standard human brain TF
preparation, which clotted human plasma in 12-13 s (arbitrarily
set as 100 units/ml).
The human
bladder carcinoma cell line J82 was obtained from the American Type
Culture Collection (Rockville, MD) and cultured in DMEM supplied with
10% FCS, L-glutamine (380 µg/ml), penicillin (100 IU/ml),
and streptomycin (100 µg/ml). For experiments approximately 2
10
cells/ml were seeded on glass coverslips and
grown for 2 days.
All transfections of COS-1 cells were done using
calcium phosphate coprecipitation procedures (19) with glycerol
shock. Cells with stably integrated constructs were selected with 500
µg/ml geneticin (G418). Total TF expression for selection of
transfectants was determined by mixing cell homogenates with human
plasma and 30 mM Ca and recording the
clotting time as described(16) . The cells were cultured in
DMEM supplied with 5% FCS, penicillin (100 IU/ml), streptomycin (100
µg/ml), and L-glutamine (380 µg/ml). For experiments
approximately 1
10
untransfected cells/ml and 2
10
transfected cells were seeded on separate glass
coverslips and grown for 2 days (the latter grew more slowly).
The
Madin-Darby Canine Kidney (MDCK) type I cell line was obtained from K.
Prydz (Oslo) and cultured in DMEM supplied with 5% FCS, L-glutamine (380 µg/ml), penicillin (100 IU/ml), and
streptomycin (100 µg/ml) in 30-mm tissue culture dishes (Nunc,
Copenhagen, Denmark). For experiments approximately 2 10
cells/ml were seeded on glass coverslips and grown for 1 day.
R is the ratio between fluorescence intensity at 345
and 385 nm excitation. R and R
signify this ratio when fura-2 is depleted of and saturated with
calcium, respectively. The calcium dissociation constant K
of fura-2 is 224 nM(21) , and
is the ratio of fluorescence between
Ca
-depleted and Ca
-saturated fura-2
at 385 nm excitation. The autofluorescence values found by measuring
fluorescence from non-fura-2-loaded cells were less than 10% of the
total fluorescence and were subtracted from the fluorescence values
before calculating the ratio.
where is the ratio of parallel changes in the fluorescence
intensities at wavelengths 345 and 385 nm (
345/
385), which
has a constant negative value independent of changes in the
fluorescence signals. By analyzing Ca
responses from
several cells with a marked and rapid rise in
[Ca
]
and without changes in
cell shape, an average value for
could be calculated. R
was measured by means of cells depleted of
Ca
by incubating them for 30 min with a
Ca
-free HSS buffer containing 1 µM 4-Br-A23187 and 5 mM EGTA. R
was
measured by incubating the same cells for another 20 min after addition
of 10 mM CaCl
.
Figure 1:
Average Ca responses
after binding of FVIIa to TF on the surface of HUVEC and J82 cells. Panel A, HUVEC incubated with IL-1
(100 units/ml) for 6 h
at 37 °C. The cells were exposed to either FVIIa (200 nM) (solid line) or HSS as control (dotted line) at 30 s
as indicated by the arrow. Panel B, HUVEC not
pretreated with IL-1
were exposed to FVIIa or HSS as in panel
A. Panel C, HUVEC pretreated with IL-1
were first
exposed to 200 nM FVIIa at 60 s and then HSS as control at 180
s (solid line) or vice versa (dotted line). Panel D, J82 cells treated like the HUVEC in panel C. n denotes the number of single cells investigated. Note
different scaling.
To see whether FVIIa
binding to TF could generate a similar response in other cell types, we
investigated the effects of FVIIa in the J82 cell line and found a
similar result (Fig. 1D). The average Ca signals following the addition of FVIIa (Fig. 1) most
frequently showed only moderate increase of
[Ca
]
(20-40 nM).
This small average response might either be due to a gradual increase
of the basal Ca
concentration in all cells or to
recruitment of single cell responses.
To distinguish between
responding and non-responding cells, an increase of more than 50 nM from the pretreated basal to the maximal Ca level was used as a criterion. All true ``responders''
will probably exceed this limit. Observing cells for 120-150 s
after agonist addition, the response rate was calculated for each well
(18-36 predetermined cells were analyzed in each well) (Fig. 2). The fraction of responding cells in
IL-1
-pretreated HUVEC exposed to 200 nM FVIIa was 21% and
in J82 cells 17%, the controls varied from 1 to 5%. By observing HUVEC
and J82 cells for a longer period, a higher response rate
(30-35%) was found due to latency of the responses. The number of
control cells responding did not increase significantly.
Figure 2:
Number of HUVEC and J82 cells responding
with Ca transients to addition of FVIIa.
Ca
transients induced by binding of FVIIa to TF were
observed for 120-150 s after addition of FVIIa (200 nM)
or HSS as control. An increase of
[Ca
]
of more than 50
nM was the criterion for a response. The response rate of each
well was calculated. The mean response rate of the wells is expressed
as percent. The bars represent standard error of the mean
(S.E.) of n different wells. Asterisks indicate a
significant difference from control (p < 0.01). The data
are from two to four different cultures.
The
Ca signals showed a heterogeneous pattern with single
transients, a train of transients (oscillations), or a spike followed
by an elevated level with superimposed oscillations (Fig. 3).
The spontaneous transients in HUVEC controls (upper panel)
reached the same maximal level as the FVIIa-induced transients (centerpanel) (265 ± 10 nM compared
to 279 ± 6 nM). In HUVEC we could thus not easily
distinguish between spontaneous and FVIIa evoked Ca
signals with regard to size of peaks, only with regard to number
of cells responding. This was the case also for the Ca
transients in J82 cells in which the maximal Ca
level reached 241 ± 5 nM, significantly lower
than in HUVEC (p < 0.001).
Figure 3:
Ca transients in single
cells. Upper panel, spontaneous Ca
transients in IL-1
-treated HUVEC after addition of HSS. Centerpanel, Ca
transients in
IL-1
-treated HUVEC after addition of FVIIa (200 nM). Lower panel, Ca
transients in J82 cells
after addition of FVIIa (200 nM). FVIIa or HSS was added at 30
s as indicated by the arrows.
FVIIa addition also increased
the basal Ca concentration. In non-responding cells
the difference between the Ca
levels at 5 and 140 s
after FVIIa addition was calculated. The basal prestimulatory
Ca
level was not used in order to avoid the effect of
possible application artifacts. When the increase of the basal
Ca
level exceeded 50 nM, the cells were
excluded, irrespective of the rate of change in Ca
levels. These data may thus represent a slight underestimate of
this part of the Ca
response. FVIIa raised basal
Ca
significantly more than in control cells both in
IL-1
-treated HUVEC and in J82 cells. In HUVEC not treated with
IL-1
, there was no significant difference between the
FVIIa-stimulated cells and controls (Table 1).
The
IL-1-treated cells had a significantly (p < 0.001)
higher basal level (132 ± 1 nM) than the untreated
HUVEC (106 ± 2 nM) suggesting that the IL-1
treatment alone increased basal Ca
levels. Of
IL-1
-pretreated HUVEC exposed to FVIIa and not responding with
Ca
transients, 18% responded with an increase of
basal Ca
of more than 15 nM, whereas only
3-6% of controls showed such a response. Similar results were
found for the J82 cells. Although the 15 nM limit was
arbitrary, these results indicated that a significant number of the
FVIIa-treated cells contributed to the increase of basal
Ca
, thus bringing the total number of responding
cells up to 40-50% according to our criteria.
To study the role of
Ca influx more directly, we then used an inhibitor of
receptor-mediated Ca
influx, SKF 96365(25) ,
that did not interfere with the FVIIa-TF interaction. The effects of
SKF 96365 were studied in both HUVEC and J82 cells and proved to be
very complex. In HUVEC Ca
influx induced by 100
µM histamine was blocked at concentrations of SKF 96365
from 30 to 50 µM (data not shown). In addition SKF 96365
induced an increase of the basal Ca
level. In
IL-1
-treated HUVEC pretreated with SKF 96365 for 3-5 min the
Ca
level was 148 ± 1 nM,
significantly higher (p < 0.001) than in controls (132
± 1 nM).
In J82 cells incubation with 30 µM SKF 96365 for 3-5 min resulted in a total inhibition of the
Ca responses normally induced by 10 µM histamine or by 1.6 µM thapsigargin, an inhibitor of
the Ca
ATPases of the intracellular Ca
stores(26) . This inhibition was not caused by inhibition
of Ca
influx since no free Ca
(in
the presence 0.5 mM EGTA) was present in the positive
controls. By direct application of 30 µM SKF 96365, we
observed a Ca
response mimicking the thapsigargin
response in Ca
-free medium (data not shown). This
effect was dose-dependent. Altogether, the SKF 96365 effects in J82
cells could be explained by a combination of inhibition of
Ca
influx and depletion of the internal
Ca
stores.
We then incubated IL-1-pretreated
HUVEC with 50 µM SKF 96365 or HSS as control for 3-5
min before addition of FVIIa. The response rates for the cells exposed
to 200 nM FVIIa were significantly higher than controls (Table 2). There was no significant difference between the
FVIIa-induced Ca
transients in HUVEC incubated with
or without SKF 96365. As expected, J82 cells incubated with 30
µM SKF for 3-5 min showed almost no responses. These
results for HUVEC support the view that Ca
influx is
not necessary for the FVIIa-induced Ca
transients.
In the same experiments the increase of the basal Ca concentration was calculated in cells that did not show a peak
response, i.e. an increase of more than 50 nM. In
both SKF 96365 and HSS-pretreated HUVEC, FVIIa induced a significantly
higher increase of the basal Ca
level than in
controls (Table 2). The lower difference between FVIIa and
control and the total higher increase in SKF 96365-pretreated cells may
be explained by a continued increase in Ca
induced by
SKF 96365. Although SKF 96365 inhibited the Ca
responses in J82 cells, there was a high and significant increase
of the basal Ca
level in the FVIIa-stimulated cells
compared to control (Table 2), indicating a rather high number of
responsive J82 cells. Since SKF 96365 inhibited Ca
influx and depleted the internal Ca
stores in
J82 cells, the increase of basal Ca
was probably
mediated by a membrane Ca
flux not inhibited by SKF
96365.
Figure 4:
Response rates in antibody pretreated J82
cells and transfected and untransfected COS-1 cells. Ca transients induced by binding of FVIIa to TF were observed for
300 s after addition of 200 nM FVIIa. J82 cells were incubated
with vWF or hTF antibodies for 30 min and then washed once. COS-1 cells
stably transfected with hTF cDNA or untransfected cells were used. An
increase of more than 50 nM was the criterion for a response.
The number of responding cells in each well was calculated and
expressed as percent of total cells. The bars represent
standard error of the mean (S.E.) of n different wells. Asterisks indicate a significant difference from control (p
0.01). The data are from two different
cultures.
COS-1 cells were stably transfected with a human TF cDNA construct giving a total TF activity of 8-10 units/mg cell protein, of which about 10% was available for interaction with FVIIa on the cell surface as revealed by the two-stage assay on intact cells. In untransfected COS-1 cells there was no detectable activity. Stimulating the hTF-transfected COS-1 cells with 200 nM FVIIa, we observed a response rate of 19% compared to 2% in untransfected COS-1 cells (p = 0.004) (Fig. 4).
Although the number of
cells responding with Ca transients clearly was
different, no significant difference in the increase of basal
Ca
levels was detected between J82 cells incubated
with the hTFab and the vWFab. In the COS-1 cells, both groups revealed
a slight increase during the experiment, but the hTF-transfected cells
had a significantly higher (p < 0.001) increase than the
untransfected cells. Altogether, these experiments show that the
effects of FVIIa in HUVEC and J82 cells depended upon available TF and
are thus mediated by the direct ligand-receptor interaction of FVIIa
and TF.
Figure 5:
FVIIa-induced Ca
oscillations in single MDCK cells. Examples of Ca
oscillations in MDCK cells treated with 10 nM (upper
panel) and 200 nM (lower panel) FVIIa at 30 s as
indicated by the arrows.
By averaging the responses of single MDCK cells at
each concentration of FVIIa, an obvious dose dependence was observed (Fig. 6). A similar dose dependence was observed when the
percentage of responding single cells was calculated (Table 3).
With control MDCK cells an average of 7% responded with spontaneous
Ca transients within 180 s after addition of vehicle,
comparable to the spontaneous response rates observed in HUVEC and J82
cells. At the physiological concentration of FVIIa in plasma (10
nM), 78% of the cells responded, and at 200 nM approximately all the MDCK cells revealed a Ca
signal.
Figure 6:
Average Ca responses
after stimulation of MDCK cells with FVIIa. MDCK cells were stimulated
with different doses (0-200 nM) of FVIIa, or HSS as
control, at 30 s as indicated by the arrow. The values were
normalized as percent of the starting basal Ca
level.
The line symbols are control (n = 118) (line
a), 2 nM (n = 96) (line b), 10
nM (n = 87) (line c), 20 nM (n = 101) (line d), 200 nM (n = 118) (line e). n is the
number of cells making up each averaged response. The results are from
three different cultures.
We then examined whether the maximal amplitude,
frequency, and latency of the Ca oscillations were
dependent on the concentration of FVIIa. Cells that responded with a
Ca
increase of more than 100 nM were
analyzed. The 100 nM level was chosen to exclude small
spontaneous transients. By calculating the difference between basal and
maximal Ca
levels, we found that the amplitude
increased with increasing FVIIa concentrations (Table 3). The
frequency was calculated for cells responding with at least three
Ca
spikes with fixed intervals after addition of
FVIIa, while the latency, i.e. the time from addition of
ligand to the beginning of the first Ca
increase was
calculated for all responding cells. When MDCK cells were observed for
180-300 s after addition of FVIIa, the frequency increased while
the latency decreased with increasing FVIIa concentrations (Table 3). Their inverse relationship was confirmed by a
significant negative correlation (r = -0.25, p < 0.05). Individual MDCK cells were able to alter their
firing frequency upon repeated additions of increasing doses of FVIIa.
When treating cells first with 20 nM and then after 180 s with
200 nM FVIIa, the mean frequency changed from 0.99 ±
0.05 to 1.50 ± 0.08 min
(n =
29). Correspondingly, the frequency increased from 0.73 ± 0.05,
via 0.87 ± 0.06 to 1.01 ± 0.07 min
when the cells were subsequently stimulated with 10, 50, and 200
nM FVIIa at a 270-300-s interval (n =
24).
The role of extracellular Ca for the
FVIIa-induced Ca
oscillations is difficult to
ascertain because the binding of ligand (FVIIa) to receptor (TF)
requires Ca
. We therefore used La
which blocks Ca
influx and which inhibits the
FVIIa
TF complex formation to a lesser degree than removal of
extracellular Ca
by EGTA addition. When treating the
cells with 200 nM FVIIa for 270 s, allowing complex formation
with TF and Ca
oscillations to start, and then adding
100 µM LaCl
, the frequency of the oscillations
decreased from 1.19 ± 0.09 to 0.92 ± 0.09
min
(n = 30, p =
0.03).
Cells that responded with a biphasic Ca signal with superimposed Ca
oscillations
changed to a simpler oscillatory pattern after addition of
La
(Fig. 7, upper panels), while
cells with base-line oscillations only decreased their frequency (lower panel). These results indicated that Ca
influx contributed to the oscillatory signal although influx was
neither necessary for generation nor maintenance of the oscillations.
However, inhibition of Ca
influx reduced their
frequency.
Figure 7:
Ca oscillations in
single cells after inhibition of Ca
influx. MDCK
cells were stimulated with 200 nM FVIIa at 30 s, and 100
µM LaCl
was added at 300 s as indicated by the arrows.
Figure 8:
Synchronized Ca
oscillations induced by FVIIa in MDCK cells. MDCK cells grown to
confluence were stimulated with 200 nM FVIIa at 30 s and then
observed for 300 s. Application of FVIIa is indicated by the arrows. Upper panel, an average Ca
response of 108 single cells in the same well. A-E indicate the time points of the pseudo-colorimages. The line below the time axis indicates the time interval of the images in Fig. 9. Lower panel, examples of three different single cell responses
in the same well. Pseudo-color image: A-E,
Ca
images taken at the points indicated in the upper panel. F, an image of the MDCK cells stained by
Colorrapid. The pseudo-color scale indicates the
Ca
concentrations.
Figure 9:
The onset of a synchronized Ca transient. The pseudo-color images show an upper right
segment of the well in Fig. 8. The 12 Ca
images are taken at a 1.2-s interval from 252.8 to 266 s as
indicated in the upper panel of Fig. 8.
We
analyzed one well in greater detail to see if ``leading''
cells could be detected that started their transients before the
others, thereby initiating a signal that established responses in their
neighboring cells. Fig. 9shows Ca images at
the start of a Ca
transient. Two or three cells seem
to increase their Ca
levels before the others and may
be such leading cells. A full sequence of Ca
images,
including several subsequent synchronous Ca
transients, indicated that some cells, especially the first cell
responding in the upper left corner of Fig. 9, started
an intercellular Ca
wave, apparently generating a
series of individual synchronous Ca
transients in
adjacent cells. These observations show that the Ca
oscillations induced by the binding of FVIIa to TF in MDCK cells
can set up a synchronous Ca
signal when the cells are
grown to confluence.
We have shown that the binding of FVIIa to TF induces a
Ca signal in four different cell types, HUVEC induced
to synthesize TF, J82 and MDCK cells constitutively expressing TF, and
COS-1 cells stably transfected with human TF cDNA. TF thus fulfills the
last requirement for being a true receptor, i.e. binding of
its ligand causes an intracellular signal. The Ca
signals were either Ca
transients produced
mainly by intracellular release of Ca
or a continuous
gradual increase of the basal Ca
concentration
generated by an altered membrane Ca
flux. The
Ca
oscillations in MDCK cells were dependent on the
concentration of FVIIa with respect to frequency, latency, maximal
amplitude, and number of responding cells, and the frequency of the
oscillations was reduced when Ca
influx was blocked
by La
. Frequency coded Ca
transients were first observed in hepatocytes (28) and
is now accepted as a general principle for hormone-induced
Ca
signals. The amplitude of the oscillations has
been reported to be constant, independent of agonist
concentrations(29) , whereas we observed increasing maximal
peak [Ca
]
levels with
increasing FVIIa concentration. The notion that these signals are
mediated by the ligand-receptor interaction of FVIIa and TF is
supported by the relationship between cellular TF activity and the
Ca
signal, i.e. no signal in uninduced HUVEC
or untransfected COS-1 cells, the dependence on external Ca
of both FVIIa-TF binding and the intracellular Ca
signal, and the blockage of the signal by a TF neutralizing
antibody. Binding sites for FVIIa other than TF have been found on
HUVEC(27) , but they have lower affinity, and our experiments
rule out these sites as possible signal transducers.
Inhibition of
Ca influx by SKF 96365 in HUVEC and the delayed
addition of EGTA in J82 cells suggest that the Ca
transients were mainly caused by release from internal
Ca
stores, although an influx component was not
excluded. We could not detect any difference between spontaneous and
FVIIa-induced transients in these cell types, indicating that they
might be generated by the same mechanisms or that FVIIa, instead of
setting up its own Ca
transients, just increased the
probability of spontaneous firing. Spontaneous Ca
transients evoked by release from internal pools have also been
reported in other non-excitable cells(30, 31) .
A
cellular Ca signal induced by hormones or other
extracellular signaling mediators is usually evoked by the activation
of phospholipase C (PLC) which hydrolyzes phosphatidylinositol (4, 5) -bisphosphate to diacylglycerol and
inositol(1, 4, 5) -trisphosphate (for review,
see (32) ). There exist two main routes for activation of PLC.
The G-protein-linked receptors activate the PLC-
isoenzyme,
whereas tyrosine kinase receptors (e.g. growth factor
receptors) or tyrosine kinase-linked receptors (e.g. T-cell
receptor, cytokine receptors) stimulate the
form of PLC. Because
of its single transmembrane domain, it seemed likely that TF induces a
Ca
signal by activation of PLC-
. TF does not
have an intrinsic tyrosine kinase activity but might be coupled to a
tyrosine kinase. However, immunoblotting using anti-phosphotyrosine
antibodies revealed no difference in the phosphorylation pattern
between unstimulated and FVIIa-stimulated cells (data not shown).
SKF 96365, one inhibitor of receptor-mediated Ca entry, has recently been shown to have some side effects. Like
thapsigargin it blocked the endosomal Ca
ATPase in
thymic lymphocytes (33) , and in HUVEC it activated a
non-selective cation channel resulting in Ca
influx(34) . Our results indicate a thapsigargin-like
effect of SKF 96365 in J82 cells, and the increased basal
Ca
level in SKF 96365 pretreated HUVEC might be
produced through the non-selective cation channel. A plausible
mechanism for the opening of this channel is by emptying of internal
Ca
stores as suggested by the capacitative
model(35) . The effect of SKF 96365 on inhibition of
Ca
influx and on emptying of Ca
stores leading to Ca
influx is therefore
probably occurring in all cell types but at different concentrations of
SKF 96365 and with different relative potency. The effects of this
inhibitor must therefore be thoroughly investigated before use in any
cell type.
The lack of inhibition of SKF 96365 on the gradual
increase of basal Ca in J82 cells indicates that the
source of this Ca
was extracellular since SKF 96365
had depleted the internal stores as witnessed by the deficient
histamine and thapsigargin responses. Neither did SKF 96365 inhibit
this phenomenon in HUVEC, suggesting that altered membrane
Ca
flux in these cells was not through the normal
receptor-operated channels inhibited by SKF 96365. Several possible
mechanisms can be responsible for such increased Ca
permeability of the plasma membrane, which has also been reported
in IFN-
-stimulated human neutrophils(36) . It might be a
potentiation of the capacitative Ca
entry as seen
with the protein kinase C activator, phorbol 12-myristate 13-acetate,
in the insulin-secreting cell line RINm5F(37) . Another
possible mechanism is decreased efflux of Ca
either
by the Ca
-ATPase or the
Na
/Ca
exchanger.
On the basis of
the mechanisms involved, Ca oscillations have been
characterized as either membrane or cytosolic oscillators (38) . Membrane oscillators arise from opening of plasma
membrane Ca
channels, either the voltage-operated
channels or the channels opened by emptying of the internal stores
(capacitative entry (35) ). Since the FVIIa-evoked
Ca
oscillations were not abolished by inhibition of
Ca
influx, they must primarily be driven by periodic
release from the internal stores, i.e. a cytosolic oscillator.
Such oscillators may be described as sinusoidal transients where the
[Ca
]
oscillates upon a
sustained elevated level or base-line transients where
[Ca
]
reaches the resting level
in between each spike. We observed both sinusoidal and base-line
oscillations. The sinusoidal ones were evoked at the maximal
concentration of FVIIa and their frequency seemed to be constant. In
neutrophils we have earlier found that sinusoidal oscillations from a
large number of cells tend to assemble around one characteristic
frequency(22) . Similar observations in lacrimal cells have
been explained by oscillating protein kinase C activity(39) .
In wells with confluent MDCK cells, we observed synchronous
Ca oscillations in 50-150 cells. Sage and
co-workers (40) reported that bradykinin induced synchronized
oscillations in monolayers of endothelial cells. Similar observations
on adjacent cells from different tissues have since been
reported(41, 42, 43) . A common feature seems
to be that these oscillations are dependent on the presence of external
Ca
or Ca
influx. Functional gap
junctions between the cells appear to be required for
synchronization(44, 45) . A membrane oscillator
controlled by electrically transmitted signals between the cells seems
to be the most likely explanation of these findings.
Another type of
co-ordinated Ca signals are intercellular
Ca
waves spreading from a single mechanically
stimulated cell(46) . Focal application of hormone, i.e. local stimulation of one single cell, induced synchronized
Ca
oscillations of pancreatic acini cells, whereas
global stimulation resulted in asynchrony (47) in contrast to
our observations. Signaling molecules diffusing through gap junctions
seem to be responsible for these phenomena with
inositol(1, 4, 5) -trisphosphate as the most
plausible candidate(48) , although waves have also been
reported to be evoked by a diffusible messenger in the absence of gap
junctions(49) . In our experiments the FVIIa-induced
Ca
oscillations were asynchronous when the MDCK cells
were not in contact with each other, whereas synchrony was observed in
confluent cell layers. This suggests that direct cell to cell contact
is required for the co-ordination of the signal. The study on
pancreatic acini (47) also revealed oscillations of higher
amplitude and increased frequency when the cells were synchronized
through gap junctions. Likewise, we observed higher spikes and
transients of shorter duration when the Ca
signal was
co-ordinated after the first transients (Fig. 8). The
synchronous oscillations that we observed conform to neither of these
types since we observed oscillations when Ca
influx
was impeded and synchrony was initiated by global application.
Consequently, our data show for the first time, to our knowledge, that
global application of agonist can induce synchronous base-line
Ca
oscillations in a confluent cell layer by periodic
release from internal stores. We speculate that this synchrony may be
co-ordinated by intercellular Ca
waves (Fig. 9) by the same mechanisms as for mechanically induced
Ca
waves and synchronous oscillations evoked by focal
application of hormone. Obviously, further investigations are required
to address this problem.
In most of our experiments, we have used a
rather high concentration of FVIIa (200 nM) compared to the
normal plasma concentration (10 nM). This was done partly
because recombinant FVIIa may have lower specific activity than
endogenous FVIIa and partly because FVIIa will bind to cell surfaces
most likely through interaction with phospholipid and proteoglycans and
thus be present at a locally increased concentration. ()If
FVIIa is able to induce a specific signal, its local concentration in vivo must exceed the normal plasma concentration which
presumably signifies the lower threshold for the cells to recognize
this signal. In HUVEC it may be more likely that the appearance of TF
triggers the signal, since FVIIa is present at all times. In MDCK cells
significant Ca
signals were found also at
concentrations (2 nM) below the normal plasma concentration.
Why FVIIa-generated Ca
transients occur in some cell
lines (HUVEC, J82, and COS-1) in only about 30% of the cells is not
known. Several possible explanations exist. Studies have shown that
whereas FVIIa/TF activity on the cell surface reaches its maximum in
about 1 min, the binding of FVIIa to TF is saturated much more
slowly(50) . This observation has not been analyzed at the
level of individual cells and could obviously be linked to
heterogeneity in cellular responses. We also observed a large variation
in the response rate in cell cultures in different wells, possibly
correlated to cell density. The Ca
responses were not
correlated to the overall TF activity of the various cell types (J82
and HUVEC showing about the same response rate but 5-10-fold
difference in TF activity), but the difference between individual cells
has not yet been analyzed. Neither did the lower response rate in HUVEC
and J82 cells compared to MDCK cells result from differences in
receptor numbers since the plasma membrane TF activity in MDCK cells
was in between the levels found in the other cell types. The more than
100-fold difference in sensitivity is therefore probably caused by a
variation in the signaling system such as a difference in the coupling
to PLC, or that the Ca
signals are evoked by partly
different mechanisms in the different cell types.
We conclude that
binding of FVIIa to TF in HUVEC, J82, and MDCK cells and hTF
cDNA-transfected COS-1 cells induces cytosolic Ca signals of different characteristics, comprising Ca
oscillations and other transients, as well as increased basal
[Ca
]
. In the MDCK cells the
oscillations are synchronous when the cells are grown to confluence,
providing the first evidence of a synchronous cytosolic Ca
oscillator generated by global application of agonist. Our work
demonstrates for the first time that TF is a true receptor which upon
binding of its ligand induces an intracellular signal. The biological
effects of these signals are now being studied. They are probably
components of a more general response chain. The role of TF in
hemostasis suggests that the FVIIa-induced signal may modulate the
coagulation cascade by regulating production and secretion of relevant
components. The fact that the TF gene is an immediate early gene and a
member of the cytokine superfamily, in addition to its signal
transducing property, suggests a role for TF also in cell biology.