Articles |
From the Gaubius Laboratory TNO-PG (Y. van den E.-S., R.E.M. de V., T.K., J.J.E.) and the Department of Cardiology, University Hospital (D.E.A.), Leiden, the Netherlands; and the Thrombosis Research Institute, London, UK (F.L.).
Correspondence to J.J. Emeis, Gaubius Laboratory TNO-PG, Zernikedreef 9, 2333 CK Leiden, the Netherlands. E-mail jj.emeis{at}pg.tno.nl
| Abstract |
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-thrombin, was almost zero after chelation of
Ca2+i, showing that an increase in
[Ca2+]i was required. It did not matter
whether the increase in [Ca2+]i came from an
intracellular or extracellular Ca2+ source.
Thrombin-induced release of TPA and vWF already started at low
[Ca2+]i, around 100 nmol/L. Half-maximal
release was found at a [Ca2+]i of 261 nmol/L
for TPA and at 222 nmol/L for vWF. The
Ca2+ signal was transduced to
calmodulin, as calmodulin
inhibitors inhibited TPA and vWF release. The
Ca2+ ionophore ionomycin dose dependently
released vWF; half-maximal vWF release occurred at a
[Ca2+]i of 311 nmol/L. In contrast, no TPA
release was found at all below a [Ca2+]i of
500 nmol/L. Thus, below 500 nmol/L [Ca2+]i,
an increase in [Ca2+]i alone was sufficient
to induce vWF release but not sufficient to induce TPA release. Protein
kinase C did not appear to be involved in TPA or vWF release, as
neither an activator nor an inhibitor of
protein kinase C significantly influenced release. Inhibition of
phospholipase A2 also did not reduce thrombin-induced TPA
and vWF release. The involvement of G proteins was studied by using
both saponin-permeabilized and intact cells. GDP-ß-S,
which inhibits heterotrimeric and small G proteins, significantly
inhibited thrombin-induced vWF and TPA release from
permeabilized cells. AlF4-,
which activates heterotrimeric G proteins, induced TPA and vWF
release in both intact and permeabilized
HUVECs. Preincubation of HUVECs with pertussis toxin
significantly inhibited thrombin-induced vWF release, due to inhibition
of thrombin-induced Ca2+ influx. Pertussis
toxin did not affect ionomycin-induced release. The
inhibitory effect of pertussis toxin was less obvious in
thrombin-induced TPA release, because it was counterbalanced by a
positive effect of the toxin on TPA release. Thus, both
inhibitory and stimulatory (pertussis toxinsensitive) G
proteins were involved in TPA release. Therefore, thrombin-induced
acute release of TPA and vWF differed in two respects. First, below a
[Ca2+]i of 500 nmol/L, an increase in
Ca2+ was sufficient for vWF release but
not for TPA release. Second, pertussis toxinsensitive G proteins were
differentially involved in acute TPA and vWF release.
Key Words: endothelial cells tissue-type plasminogen activator von Willebrand factor calcium G proteins
| Introduction |
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In this study, we investigated the role of second messenger systems in acute TPA and vWF release from cultured human endothelial cells. To this end, we used a system with HUVECs, which shows acute release characteristics comparable to the situation in vivo and which is suitable to study both TPA and vWF release.1 12 Thrombin was used as a stimulus to induce acute release, since it is a potent and physiologically important stimulus for acute TPA and vWF release.13 14
It is known that the addition of thrombin rapidly increases the [Ca2+]i in endothelial cells.15 Furthermore, thrombin and Ca2+ ionophore, which both increase [Ca2+]i, stimulate vWF release from HUVECs in vitro,4 5 6 7 8 9 as does the introduction of Ca2+ into permeabilized cells.7 16 It is well established that next to Ca2+, G proteins are also involved in secretion.17 18 19 G proteins, including one or more PTX-sensitive G proteins, are involved in transducing (thrombin) receptor-mediated signals in endothelial cells20 21 22 23 ; the latter G proteins include one or more pertussis toxinsensitive G proteins.23 Since only little is known about the involvement of Ca2+ in TPA release24 25 or about the involvement of G proteins in TPA and vWF release,16 we investigated in this study the role of Ca2+ and G proteins in acute release (regulated secretion) of TPA and vWF from HUVECs.
We demonstrated that Ca2+ was essential for thrombin-induced TPA and vWF release and that it most likely acted via calmodulin. At comparable levels of [Ca2+]i, thrombin induced release of TPA and of vWF to the same extent. On stimulation with Ca2+ ionophore, however, TPA release required higher levels of [Ca2+]i than did vWF release. In experiments involving GDP-ß-S, AlF4- (AlCl3 plus NaF), and PTX, PTX-sensitive G proteins were found to be differentially involved in the acute release of TPA and vWF.
| Methods |
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-thrombin,
sodium butyrate, cyclopiazonic acid, TFP,
glutaraldehyde, indomethacin, PMA, LDH
reagent kit, AlF4-, saponin, PTX, and
GDP-ß-S were from Sigma Chemical Company. W-7, ETYA, and SK&F96365
were bought from Biomol. Calmidazolium came from
Janssen Pharmaceutica. Fura 2-AM and BAPTA-AM were bought from
Molecular Probes, ionomycin from Calbiochem, and EGTA from Aldrich.
Materials used in the vWF and TPA ELISAs have been
described.25 26 All other chemicals were from Merck.
AACOCF3 was a gift from Dr G. Nalbone
(Laboratoire d'Hématologie, Marseille, France). Ro-31-8220 was a
gift from Dr G. Lawton (Hoffmann La Roche, Welwyn Garden City,
UK).
Cell Culture
HUVECs were isolated by using the method of Jaffe et
al27 and cultured as previously described28
in total M199 (medium 199 supplemented with 10% [vol/vol]
human serum, 10% [vol/vol] heat-inactivated
newborn calf serum, 100 IU/mL penicillin, 100 µg/mL
streptomycin, 200 µg/mL endothelial cell
growth factor, 2.5 U/mL heparin, and 2 mmol/L
L-glutamine), under 5% CO2 at 37°C. Cells
were passaged once by trypsin/EDTA treatment at a split ratio of 1:3
and cultured to confluence in 2-cm2 wells (final density
2x105 cells per well) or on 1.44-cm2 glass
coverslips that had been coated with glutaraldehyde
cross-linked gelatin.
Induction of Acute Release of TPA and vWF
Acute release of TPA and vWF was studied as previously
described.12 In short, first-passage confluent HUVECs were
incubated for 24 hours before an experiment in total M199 containing
1 mmol/L butyrate.29 Subsequently, HUVECs were
incubated in medium 199 containing 20% (vol/vol) newborn calf
serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, and
2 mmol/L L-glutamine for 2 hours before an
experiment, and the conditioned media were collected. The cells were
then washed with sterile PBS, and 0.3 mL M199/HSA (medium 199
containing 0.03% [wt/vol] HSA, 100 IU/mL penicillin, 100
µg/mL streptomycin, and 2 mmol/L
L-glutamine) was added to the cells. Inhibitors
were diluted in M199/HSA and added during this time period, as
indicated. Thirty minutes later, the appropriate stimulus in M199/HSA
or vehicle was added to the cells and the media were collected after 3
minutes. In this study, only those cell cultures that released TPA on
stimulation are reported (for unknown reasons, 10% to 15% of HUVEC
cultures do not release detectable amounts of TPA on stimulation). When
an experiment involving Ca2+ was performed, M199 was
replaced by Tyrode's solution (composition in mmol/L: NaCl
132, KCl 4, MgCl2 1, NaHCO3 11.9,
NaH2PO4 0.36, D-glucose 10, HEPES
12.25, and CaCl2 1.6 or EGTA 0.1) supplemented with
vitamins and amino acids, starting at the 2-hour preincubation period.
Before the induction of acute release, cells were incubated with
Tyrode's/HSA (Tyrode's solution containing 0.03% [wt/vol] HSA, 100
IU/mL penicillin, 100 µg/mL streptomycin, and 2
mmol/L L-glutamine) for 10 minutes. If required,
10 µmol/L BAPTA-AM was present during the last hour
of the preincubation period. All incubations were done at 37°C.
Assays for TPA and vWF were always done on the same conditioned
media.
Minimal Cell Permeabilization
First-passage HUVECs were cultured as described above and
permeabilized essentially as described by Birch et
al.7 The cells were washed with sterile PBS and then
incubated for 10 minutes with permeabilization buffer (composition
in mmol/L: NaCl 20, KCl 100, MgCl2 1, HEPES 30,
EGTA 0.1, L-glutamine 2, gelatin 0.1% [wt/vol], HSA
0.03% [wt/vol], pH 7.0) containing 15 µg/mL saponin. The
[Ca2+]e in this buffer was 100 nmol/L
(from impurities). A single stock solution of saponin (20 mg/mL
in permeabilization buffer) was used in all experiments described.
Saponin was diluted in a strictly standardized way and prewarmed to
37°C for 45 minutes before each experiment. Cells were stimulated for
3 minutes with I-thrombin after a 10-minute incubation with
permeabilization buffer.
Intracellular Free Ca2+ Measurements
First-passage HUVECs cultured on coverslips were loaded with
4 µmol/L fura 2-AM in M199/HSA for 45 minutes at 37°C.
After the loading period, the cells were washed twice with PBS and put
immediately in a coverslip holder, which was then placed into a cuvette
containing Tyrode's solution. The cuvette was introduced into a
thermostatically controlled chamber at 37°C in a Perkin-Elmer LS-5B
single-wavelength fluorescence spectrophotometer. The
excitation wavelength was alternated between 338 and 380 nm, and
emitted light was collected at 495 nm. Slits were set at the 10.0
setting for excitation and emission. In each experiment, background
autofluorescence was determined and subtracted. Calibration and
calculations were performed as described by Grynkiewicz et
al.30 The maximal fluorescence ratio was
determined by addition of 1 or 2 µmol/L ionomycin, and
the minimal fluorescence ratio was determined after subsequent
incubation with 10 mmol/L EGTA in 1 mol/L Tris, pH
8.5, for 15 minutes.
Use of PTX, GDP-ß-S, and AlF4-
PTX, which inactivates
Gi/o,31 32 was included in the culture medium
during both the 2-hour and the 30-minute preincubation periods. A
concentration of 1 µg/mL PTX was used, since that
concentration fully inactivates
Gi/o.33 GDP-ß-S (2 mmol/L), an
inhibitor of both small and heterotrimeric G proteins, was
present during the 10 minutes of permeabilization and also during
the 3 minutes of thrombin stimulation. AlF4-
(20 µmol/L AlCl3 plus 30 mmol/L
NaF), which stimulates heterotrimeric G proteins,34 was
dissolved in permeabilization buffer to prevent formation of
CaF2 and Al2(PO4)3.
Assays
Human TPA antigen was measured by ELISA as
described.26 Recombinant human one-chain TPA (activase)
was used for calibration. The detection limit in this assay is 10
pg/mL. vWF antigen was measured by ELISA as
described.35 Human pooled plasma (0.078% to 1.25%) was
used for calibration. The vWF concentration is given as units per
milliliter, 100 U being defined as the amount of vWF antigen
present in 1 mL of pooled human plasma.
6-Ketoprostaglandin F1
was measured by
radioimmunoassay by Dr F.J. Zijlstra (Erasmus University, Rotterdam,
the Netherlands).
Data Presentation, Units Used, Definitions, and
Statistics
Acute release is defined as the concentration of TPA (or vWF) in
medium with stimulus minus the concentration in medium without stimulus
at t=3 minutes. The amount of acute release is expressed as a
percentage (±SD) of the acute release found in thrombin-treated cells
(determined in at least three independent experiments performed in
triplicate). Constitutive secretion is defined as the amount of protein
secreted in Tyrode's/HSA or M199/HSA during the 10- or 30-minute
preincubation period. Changes in the amount of protein secreted during
this preincubation period as a result of using inhibitors
may be due to altered constitutive secretion or to regulated secretion.
Such changes will be indicated, but they were not further investigated.
[Ca2+]i was measured in duplicate, while TPA
and vWF release were measured in parallel in triplicate cups.
All data shown are mean±SD (number of determinations), or median (95% CI). Statistical significance was determined by comparing treated cells with control cells by one-way ANOVA, followed by Bonferroni's modified t test. Experiments involving dose-response curves in the presence or absence of an inhibitor were analyzed by two-way ANOVA. Differences are considered significant at P<.05 (two sided).
| Results |
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Increase in [Ca2+]i and Release of TPA
and vWF by Thrombin
The system described was then used in combination with the
Ca2+ dye fura 2-AM to investigate the involvement of
Ca2+ in the acute release of TPA and vWF (Fig 1A
to
1C). Under basal conditions, the
[Ca2+]i was 76±36 nmol/L (n=10) in
first-passage HUVECs. When cells were stimulated with 1 NIH U/mL of
thrombin, [Ca2+]i increased in less than 1
minute to a peak level of 792±187 nmol/L (n=10). Thereafter,
[Ca2+]i decreased to a plateau level of
224±218 nmol/L (n=10). The peak
[Ca2+]i induced by thrombin preceded in time
the acute release of TPA and vWF: these latter processes are maximal at
1 and 3 minutes, respectively.12
|
Role of Ca2+-Calmodulin in Thrombin-Induced
TPA and vWF Release
To study whether the increase in [Ca2+]i
is essential in thrombin-induced TPA and vWF release,
[Ca2+]i was minimized by incubating the cells
with the cell-permeable Ca2+ chelator BAPTA-AM in the
absence of Ca2+e. Under these conditions, no
increase in [Ca2+]i was found on addition of
thrombin (Fig 1A
). The acute release of TPA and vWF, measured in
parallel, was almost zero (Table 1
).
|
To study whether Ca2+ influx contributed to acute release
of TPA and vWF, HUVECs were incubated for 10 minutes before thrombin
treatment with either 50 µmol/L SK&F96365 (an
inhibitor of Ca2+ influx)36 or
Ca2+-free Tyrode's solution containing 100
µmol/L EGTA. The peak level of
[Ca2+]i was slightly reduced, and
[Ca2+]i rapidly returned to basal levels in
Tyrode's solution containing SK&F96365, as well as in
Ca2+-free Tyrode's solution containing 100
µmol/L EGTA (Fig 1B
). However, TPA and vWF release were only
partially inhibited under these conditions (Table 1
).
The contribution of Ca2+i pools was studied as
follows: 10 µmol/L cyclopiazonic
acid37 was used to empty the Ca2+i
pools, in the absence of Ca2+e. On addition of
cyclopiazonic acid in Ca2+-free Tyrode's
solution, a small transient increase in
[Ca2+]i was seen. At 10 minutes after
cyclopiazonic acid addition, thrombin induced no further
increase in [Ca2+]i (Fig 1C
). TPA and vWF
release were diminished to 41% and 60%, respectively, under these
conditions (Table 1
; see also "Discussion").
Cyclopiazonic acid did not change the constitutive
secretion of TPA or vWF in the absence of
Ca2+e.
Together, these experiments suggested that an increase in [Ca2+]i was necessary for thrombin-induced acute release of TPA and vWF but that it was not important from which source the Ca2+ was derived.
To answer the question as to whether Ca2+ acted via binding
to calmodulin, HUVECs were preincubated for 30 minutes with
various concentrations of the calmodulin
inhibitor TFP, calmidazolium, or W-7
before stimulation with
-thrombin. All inhibitors
inhibited TPA and vWF release dose dependently (not shown). At optimal
concentrations, all three inhibitors significantly
inhibited the release of TPA and vWF (Table 2
), suggesting that the increase in
[Ca2+]i is transduced to
calmodulin. TFP and W-7 did not significantly affect
constitutive secretion during the preincubation period.
Calmidazolium, however, significantly enhanced vWF
secretion during the preincubation period; a similar effect has been
observed in vivo in a perfused rat hindleg system.25
|
Increase in [Ca2+]i Necessary for
Thrombin-Induced TPA and vWF Release
The Ca2+i levels and the acute release of
TPA and vWF were measured in parallel after stimulation with increasing
concentrations of thrombin. In Fig 2
, the
peak concentration [Ca2+]i is plotted against
TPA or vWF release (expressed as percentage of the release induced by 1
NIH U/mL of thrombin). Acute release of TPA and vWF started around 100
nmol/L [Ca2+]i and increased very
rapidly when [Ca2+]i increased further. By
linear regression analysis, it was calculated that 50% TPA
release occurred at 269 nmol/L Ca2+ (95% CI, 211 to
338 nmol/L) and 50% vWF release at 222 nmol/L (95% CI,
182 to 279 nmol/L; see legend to Fig 2
). In combination with the
fact that complete removal of [Ca2+]i in
HUVECs with BAPTA-AM in the absence of Ca2+e
inhibited thrombin-induced TPA and vWF release (see Table 1
), these
data suggested that an increase in [Ca2+]i
above 100 nmol/L was necessary for thrombin-induced acute
release of TPA and vWF to occur.
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Effect of Ionomycin on TPA and vWF Release
To study whether an increase in
[Ca2+]i was sufficient to induce acute
release of TPA and vWF, we used increasing doses (up to 0.9
µmol/L) of the Ca2+ ionophore ionomycin to
increase [Ca2+]i stepwise. The amount of TPA
and vWF acutely released by ionomycin (expressed as percentage of the
release induced by 1 NIH U/mL of
-thrombin) is plotted against
[Ca2+]i in Fig 3A
for TPA and 3B for vWF. The results
showed that increases in [Ca2+]i induced by
ionomycin dose dependently induced acute release of vWF (Fig 3B
). The
[Ca2+]i needed for 50% vWF release by
ionomycin was 311 nmol/L (95% CI, 233 to 424 nmol/L),
which is slightly higher than the [Ca2+]i
needed for 50% vWF release induced by thrombin (222 nmol/L). In
contrast, ionomycin did not induce any TPA release below 500
nmol/L [Ca2+]i (Fig 3A
). Together,
these data showed that ionomycin did release vWF but was unable to
release TPA as long as [Ca2+]i remained below
500 nmol/L. Thus, thrombin may activate, besides
Ca2+, other regulatory mechanisms required for TPA release
but not for vWF release.
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Involvement of PKC in Thrombin-Induced TPA Release
Because thrombin activates PKC in
HUVECs,38 we investigated whether PKC was involved in
thrombin-stimulated TPA release. To this end, the cells were
preincubated for 30 minutes with the PKC inhibitor
Ro-31-8220, followed by induction of acute release with 1 NIH U/mL
-thrombin. Ro-31-8220 increased thrombin-induced TPA and vWF release
slightly, but not significantly (not shown).
It proved not possible to study the role of PKC by downregulating
PKC activity by PMA, since no TPA storage pool was left after 24 hours
of treatment with 1 µmol/L PMA. This finding is
comparable to that of the vWF storage pool, which is also absent after
24 hours of PMA treatment.39 Next, we investigated whether
activation of PKC by PMA could improve the poor TPA release induced by
ionomycin at below 500 nmol/L [Ca2+]i.
HUVECs were stimulated with increasing doses of the Ca2+
ionophore ionomycin in the presence or absence of 100 nmol/L PMA
(which itself did not induce any TPA release during the 3-minute
stimulation period). PMA had no effect on ionomycin-induced TPA release
below 500 nmol/L [Ca2+]i, whereas it
slightly, but not significantly, enhanced the ionomycin-induced TPA
release above 500 nmol/L [Ca2+]i. This
slight effect of PMA on ionomycin-induced TPA release is not mediated
by an effect of PMA on [Ca2+]i in HUVECs (see
Reference 3838 and personal communication from R. Draaijer, July 1996).
By Western blot analysis (not shown), we found only the
PMA-sensitive PKC isotypes
, ß, and
in our first-passage
endothelial cells, but not the isotypes
,
, and
I (in agreement with published data40 ), which makes it
unlikely that thrombin would influence release via PMA-insensitive PKC
isotypes.
Involvement of Eicosanoids in Thrombin-Induced TPA Release
Thrombin will also activate PLA2 in
HUVECs.16 19 23 However, neither the combined
lipoxygenase/cyclooxygenase
inhibitor ETYA41 (25 µmol/L),
the cyclooxygenase inhibitor
indomethacin41 (25 µmol/L),
nor the PLA2 inhibitor AACOCF342
(10 µmol/L) caused any inhibition of TPA or vWF release
(not shown). As the inhibitors fully (ETYA,
indomethacin) or significantly (for 70%, AACOC3)
inhibited the thrombin-induced production of
6-ketoprostaglandin F1
, these experiments
indicated that eicosanoids did not play a role in thrombin-induced
release.
Involvement of G Proteins in Thrombin-Induced TPA Release
Since thrombin activates G proteins in
HUVECs20 21 22 23 and because G proteins play a role in
exocytosis,17 18 19 we studied the involvement of G proteins
in thrombin-stimulated acute release. To be able to use
cell-impermeable or poorly cell-permeable compounds such as GDP-ß-S
and AlF4-, which are necessary to be able to
study G protein involvement, we used permeabilized
HUVECs. To this end, we chose the system described by Birch et
al,7 in which HUVECs are minimally
permeabilized by saponin, because in this system,
exocytosis of at least vWF was shown to be
maintained.7
Release From Minimally Permeabilized Cells
In HUVECs minimally permeabilized by using
saponin,7 the amounts of vWF and TPA released into the
permeabilization medium during the 10-minute permeabilization period
were increased by about 50% compared with cells incubated without
saponin (54±25% for vWF and 54±36% for TPA, respectively; n=6).
Thrombin dose dependently released vWF (Fig 4A
) and TPA (Fig 4B
) from
saponin-permeabilized cells. The release induced by 1
NIH U/mL human
-thrombin was, in permeabilized
cells, very similar to the release induced by thrombin in
nonpermeabilized cells (114±12% for vWF and 89±16%
for TPA, respectively; n=6). The release was not due to cell lysis, as
the amount of LDH in the medium was only 5±3% of that present in
the cell extracts. To further check the integrity of our cells,
permeabilized cells were preincubated with the
calmodulin inhibitor TFP to verify whether the
acute release of vWF and TPA was still mediated by
calmodulin. Acute thrombin-induced release from
permeabilized cells was inhibited by TFP by 83±9% for
vWF and 100±0% for TPA (both n=3). Thus, thrombin-induced release
from minimally saponin-permeabilized HUVECs was very
similar to thrombin-induced release from intact cells.
|
G Proteins in Thrombin-Induced vWF and TPA Release
Since both heterotrimeric and small G proteins might be
involved in thrombin-induced vWF and TPA release, the effect of
GDP-ß-S (a stable GDP analogue which inactivates both
types of G proteins) on thrombin-induced vWF and TPA release was
studied first. When HUVECs had been preincubated for 10 minutes with
2 mmol/L GDP-ß-S, the thrombin-induced vWF and TPA
release was inhibited completely up to 0.03 NIH U/mL
-thrombin and
significantly by about 30% above this concentration (Fig 4A
and 4B
).
To discriminate between the role of heterotrimeric G proteins and small G proteins, HUVECs were stimulated with AlF4-, which activates heterotrimeric but not small G proteins.34 AlF4- induced vWF and TPA release dose dependently (not shown). At 30 mmol/L fluoride, AlF4- induced (compared with thrombin treatment) 21±19% vWF release in intact cells and 79±43% in permeabilized cells. Release of TPA induced by AlF4- was 81±31% in intact cells and 121±22% in permeabilized cells, respectively, compared with thrombin treatment (n=3). Together, these data suggested that at least heterotrimeric G proteins were involved in vWF and TPA release.
Effect of PTX on vWF Release
To further define the classes of heterotrimeric G proteins
involved in thrombin-induced release, HUVECs were incubated
for 2.5 hours with 1 µg/mL PTX, which inactivates
heterotrimeric G proteins of the classes
Gi/o.33 Pretreatment with PTX did not change
the constitutive secretion of vWF, but thrombin-induced vWF release was
significantly inhibited by PTX at
-thrombin concentrations
0.03 NIH U/mL (Fig 5A
) and maximally by
29% at 0.3 NIH U/mL of
-thrombin. Pretreatment with PTX reduced the
fluorescence ratio of the peak Ca2+ phase and
(significantly) the fluorescence ratio of the plateau
Ca2+ phase (Table 3
),
supporting the hypothesis that PTX inhibited thrombin-induced
Ca2+ influx into HUVECs.
|
|
To test the hypothesis that PTX exerted its action on vWF release
via Ca2+ influx, we induced acute release of vWF with
various concentrations of
-thrombin in Ca2+-free
Tyrode's solution. No effect of PTX on release was seen under these
conditions (Fig 5B
). Pretreatment with PTX did not affect vWF release
induced by ionomycin either (Fig 5C
). The effect of PTX on
thrombin-induced vWF thus was not seen when
[Ca2+]i was increased receptor independently
or when there was no Ca2+ influx.
Effects of PTX on TPA Release
PTX did not affect constitutive TPA secretion but inhibited
thrombin-induced TPA release (maximally 20% at 0.1 NIH U/mL; Fig 6A
). The effect of PTX on
thrombin-induced TPA release was, however, not significant (by two-way
ANOVA), possibly because the inhibition by PTX was less at lower and
higher concentrations of
-thrombin, and no inhibition was found at 1
NIH U/mL
-thrombin. These data showed that the effects of PTX on TPA
release were more complex than on vWF release. In contrast to vWF
release (Fig 5B
), PTX significantly enhanced TPA release in
Ca2+-free Tyrode's solution (Fig 6B
). Pretreatment with
PTX also slightly, but not significantly, enhanced TPA release by
ionomycin (Fig 6C
), maximally to 132±12% (n=3) at 1
µmol/L ionomycin.
|
| Discussion |
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In the present paper, we studied the role of Ca2+ and G proteins in thrombin-stimulated TPA and vWF release. An increase in [Ca2+]i was essential for TPA and vWF release, as complete chelation of [Ca2+]i with BAPTA-AM in Ca2+-free Tyrode's solution almost completely inhibited both TPA and vWF release. Since preincubation with three different calmodulin inhibitors (TFP, W-7, and calmidazolium) significantly inhibited both TPA and vWF release, Ca2+ most probably transduced to the Ca2+-binding protein calmodulin. These data are in agreement with data from Birch et al,9 who showed that vWF release induced by thrombin was almost nil after incubation with EGTA and MAPTA-AM (a Ca2+-chelating agent similar to BAPTA-AM) and that introduction into permeabilized HUVECs of a calmodulin-inhibitory peptide inhibited vWF release.9 Only the observation that despite the absence of an increase in [Ca2+]i on thrombin stimulation, TPA and vWF release still occurred in cells treated with cyclopiazonic acid in the absence of Ca2+e was unexpected. However, under the latter conditions, [Ca2+]i was not chelated as when BAPTA-AM was used. Possibly, the early transient increase in [Ca2+]i induced by cyclopiazonic acid might have contributed later to TPA and vWF release. Another possibility is that local increases in [Ca2+]i (not detectable in our system) might still have occurred and induced TPA and vWF release.
Furthermore, our data showed that inhibition of Ca2+ influx
or depletion of the Ca2+i pools inhibited TPA
and vWF release only slightly (Table 1
). Under these conditions, a
reduced [Ca2+]i peak was seen on the addition
of thrombin (Fig 1
). Because the peak [Ca2+]i
strongly correlated with the extent of TPA and vWF release (Fig 2
), it
is likely that depletion of the Ca2+i pool or
inhibition of Ca2+ influx affected TPA and vWF release via
an effect on peak [Ca2+]i. Apparently, the
source from which the increase in [Ca2+]i was
derived was irrelevant (Table 1
). These data are in contrast to
experiments in a rat perfused hindleg system, in which depletion of
Ca2+e with EDTA after a few minutes fully
inhibited TPA and vWF release.25 43 This difference might
be due to the fact that the Ca2+i pool is, in
vivo, smaller than in vitro44 and more rapidly depleted.
We therefore suggest that Ca2+ influx plays a more
prominent role in increasing [Ca2+]i and
hence also in TPA and vWF release in vivo than in vitro. The same may
be true for other endothelial cell systems in which
activation depends on an increase in
[Ca2+]i.
Discrepancies exist in the literature regarding the role of Ca2+ influx in vWF release from HUVECs. In some studies,4 5 7 chelation of Ca2+e with an excess of EDTA or EGTA for at least 10 minutes inhibited vWF release. However, Birch et al,9 who used Ca2+-free buffers (not containing large amounts of EDTA or EGTA), did not find Ca2+ influx to be essential for thrombin-stimulated vWF release. In our experiments, Ca2+-free Tyrode's solution with a low concentration (0.1 mmol/L) of EGTA was used. We suggest that incubation of HUVECs with large amounts of EDTA and/or EGTA for a prolonged period of time not only removes Ca2+e but also decreases the amount of Ca2+ in the intracellular storage pools sufficiently to reduce the peak [Ca2+]i after stimulation and thus to reduce release.
The amount of [Ca2+]i needed for 50% vWF release induced by ionomycin was 311 nmol/L, comparable to a [Ca2+]i of 222 nmol/L needed for 50% vWF release induced by thrombin and also comparable to the [Ca2+]i of 350 nmol/L needed for 50% vWF release induced by thrombin, as found by Birch et al.9 Because thrombin and ionomycin induced 50% vWF release at comparable levels of [Ca2+]i, we suggest that Ca2+ alone is sufficient to induce acute vWF release, a conclusion also reached by Frearson et al.16 Carew et al8 noted that ATP increased [Ca2+]i in HUVECs but did not induce vWF secretion. This result might have occurred because these authors measured vWF release only after 60 minutes, while, due to ATP breakdown by extracellular ATPases, the increase in [Ca2+]i may not have been sustained long enough to have a measurable effect on the final vWF level over this long period. Another explanation might be that in contrast to most other Ca2+-increasing agents, ATP also increases cAMP in HUVECs.45
Our ionomycin experiments showed different results for TPA release than for vWF release. While thrombin-induced TPA release occurred at all [Ca2+]i >100 nmol/L, ionomycin-induced release of TPA was only seen if Ca2+ levels increased to >500 nmol/L. In contrast, vWF release occurred in all instances with [Ca2+]i >100 nmol/L. We have no explanation for the sudden occurrence of ionomycin-induced TPA release above 500 nmol/L [Ca2+]i.
Calmidazolium significantly increased vWF secretion
during the preincubation period. Calmidazolium has
been reported46 to increase
[Ca2+]i in bovine aortic
endothelial cells, an observation confirmed by us for
HUVECs (our unpublished data, 1996). This increase in
[Ca2+]i likely induced acute vWF release
during the preincubation period, which would explain the reduced
subsequent thrombin-induced release of vWF (Table 2
). Interestingly,
calmidazolium did not increase TPA secretion during
the preincubation period and in this respect resembled ionomycin. This
finding suggests again that at low Ca2+ levels, an increase
in [Ca2+]i is not sufficient to induce TPA
secretion.
Since activation of the thrombin receptor also activates (in HUVECs) PLA2 and PKC,16 19 23 38 we studied their role in TPA and vWF release. The PKC inhibitor Ro-31-8220 did not significantly affect thrombin-induced TPA and vWF release, while PMA did not induce acute TPA or vWF release (over 3 minutes). Our data concerning vWF release agree with results from other groups, who found that thrombin-induced vWF release is independent of PKC activation.7 8 Carew et al8 also found a small increase in thrombin-induced vWF release after preincubation with 0.1 µmol/L Ro-31-8220. Activation of PKC by PMA modulated ionomycin-induced TPA release but slightly, and only at high levels of [Ca2+]i. We therefore conclude that PKC is not essential for acute (eg, within 3 minutes) thrombin-induced TPA and vWF release. However, PMA is known to induce delayed, low-level, and long-lasting vWF release, which is maximal in HUVECs after 30 minutes.47 This "slow" vWF release has also been found in the rat perfused hindleg model.25 This type of slow release might explain the effect of PMA on Ca2+ ionophoreinduced vWF release found by Carew et al.8
Indomethacin, ETYA, and AACOCF3 significantly inhibited prostacyclin synthesis without affecting TPA or vWF release. We thus consider it highly unlikely that metabolites of the eicosanoid pathway are involved in thrombin-mediated TPA or vWF release.
An important role has been ascribed to G proteins in exocytotic
processes as such18 and in transducing (thrombin)
receptor-mediated signals in the endothelial
cell.18 19 20 21 22 23 48 An involvement of one or more heterotrimeric
G proteins in acute release from HUVECs was suggested by the
experiments with AlF4-, which released TPA and
vWF from intact cells, and to an even greater extent from
permeabilized cells. It is likely that
AlF4- stimulated the heterotrimeric G proteins
involved in release more easily in permeabilized cells
than in intact cells because the latter are more accessible. Such
findings have, for instance, been reported by Inoue et
al,49 who showed that AlF4-
activated Gi but not Gs in intact
cells, but that AlF4- activated both G
proteins in cell membrane preparations.49 The 30%
inhibition of thrombin-induced release by GDP-ß-S (Fig 4
) also
suggested that G proteins were involved in TPA and vWF secretion.
Pretreatment of HUVECs with PTX inhibited vWF release by about 30% at
all concentrations of
-thrombin tested, most likely due to
inhibition of Ca2+ influx (Fig 5
and Table 3
). The
mechanism by which a PTX-sensitive G protein mediates Ca2+
influx in HUVECs is still unknown. Possibly, a PTX-sensitive G protein
that is coupled to the thrombin receptor activates, either
directly or indirectly, a Ca2+ influx channel. Such a
mechanism has been described by Graier et al,50 who showed
that an increase in 5,6-epoxyeicosatrienoic acid induced
Ca2+ influx into HUVECs. 5,6-Epoxyeicosatrienoic acid is
formed out of arachidonic acid, liberated from membrane
lipids by activated PLA2, by
monooxygenation. However, in our system, neither
inhibition of PLA2 by AACOCF3 nor inhibition of
monooxygenation by ETYA inhibited thrombin-induced
Ca2+ influx, making this explanation unlikely. The initial
rise in [Ca2+]i due to activation of
phospholipase C and the formation of IP3 was slightly,
though not significantly, inhibited by PTX (Table 3
). This observation
is in agreement with results from Garcia et al,33 who also
showed that the thrombin-induced initial increase in
[Ca2+]i is PTX insensitive. Other effects of
thrombin, eg, the activation of c-fos,23 may,
however, be PTX sensitive. Also, other receptors may be coupled in
HUVECs to phospholipase C via PTX-sensitive G proteins, as shown for
ATP51 and histamine.21
Our data suggest that a second PTX-sensitive G protein may additionally
be involved in acute release of TPA specifically. The effect of this G
protein, which reduces TPA release, is usually masked by the effect of
the G protein modulating Ca2+ influx. Its effect could be
observed when HUVECs were stimulated with thrombin under
Ca2+-free conditions (Fig 6B
) or when
[Ca2+]i was increased receptor independently
by using ionomycin (Fig 6C
). Although the effects on thrombin- and
ionomycin-induced secretion were not significant, but only suggestive,
these data (Fig 6A
through 6C) may suggest that the
inhibitory G protein acted distally to the increase in
[Ca2+]i.52 This observation is
comparable to those in chromaffin cells, in which the regulated
secretion of catecholamines induced by various
concentrations of Ca2+ is also enhanced by pretreatment
with PTX.53 The inhibitory G protein in
chromaffin cells is characterized as a Go protein and is
located on the chromaffin granule membrane.54 The
activation of this Go is mediated via GAP43, a cytoplasmic
pseudoreceptor, which is sensitive to PKC and
calmodulin.55 It has been proposed that this
Go is continuously activated by basal levels of
Ca2+ and thus prevents regulated secretion under
nonstimulated conditions. It is possible that the
inhibitory G protein in our cells is also continuously
activated by basal levels of Ca2+ and thus prevents
the acute release of TPA under nonstimulated conditions.
In summary, our results showed that acute release of both TPA and vWF requires an increase in [Ca2+]i above 100 nmol/L. Such an increase is sufficient to cause the acute release of vWF but (below 500 nmol/L) not sufficient to cause release of TPA. A PTX-sensitive G protein was involved in thrombin-induced Ca2+ influx. A second PTX-sensitive G protein is likely involved in increasing the acute release of TPA but not vWF.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
|---|
Received June 30, 1996; accepted December 19, 1996.
| References |
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and ß protein
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