Articles |
From Zentrum der Physiologie, Klinikum der JWG-Universität, Frankfurt/Main, Germany.
Correspondence to Dr Ingrid Fleming, Zentrum der Physiologie, Klinikum der JWG-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.
| Abstract |
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42
and
44 kD). These proteins were identified by immunoprecipitation as
the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP
kinase). The agonist-induced tyrosine phosphorylation of the 42-/44-kD
doublet was sensitive to the tyrosine kinase inhibitors genistein (100
µmol/L) and piceatannol (10 µmol/L) and was inhibited by the
removal of Ca2+ from the extracellular medium. In
fura 2loaded endothelial cells, inhibition of tyrosine kinases
attenuated Ca2+ signaling after stimulation with
either bradykinin (30 nmol/L) or thapsigargin (30 nmol/L). Since
inhibition of tyrosine kinases specifically attenuates the plateau
phase of the Ca2+ response after stimulation, the
effect of tyrosine kinase inhibition appeared to be mostly associated
with the influx of Ca2+ from the extracellular
space. These data demonstrate that the signal transduction cascade
initiated by receptor-dependent and -independent stimulation of
endothelial cells includes the following: a tyrosine kinase
inhibitorsensitive transmembranous influx of Ca2+
and the tyrosine phosphorylation of two cytosolic protein substrates
identified as MAP kinases. Furthermore, on one hand, an increase in
[Ca2+]i was essential for tyrosine
phosphorylation; on the other, the Ca2+ influx was
modulated by tyrosine phosphorylation. This finding documents the
mutual dependence of these two crucial signaling pathways in
endothelial cells.
Key Words: endothelial cells mitogen-activated protein kinase intracellular Ca2+ genistein
| Introduction |
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| Materials and Methods |
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-nitro-L-arginine and HEPES,
from Serva; medium 119 (M-119), from GIBCO BRL; and penicillin,
streptomycin, L-glutamine, glutathione, and
L(+)-ascorbic acid (Biotect protection medium), from
Biochrom. Bradykinin and all other substances were obtained from Sigma
Chemical Co.
Cell Culture
Human umbilical vein endothelial cells, isolated from umbilical
cords as previously described,10 were seeded either on
glass coverslips (Bachofer) or in culture dishes (35 mm, Falcon)
containing M-119 and 20% heat-inactivated fetal calf serum (Vitromex)
supplemented with penicillin (50 U/mL), streptomycin (50 µg/mL),
L-glutamine (1 mmol/L), glutathione (5 mg/mL), and
L(+)-ascorbic acid (5 mg/mL).
[Ca2+]i was estimated in cells grown
on coverslips for 24 hours.
Immunoblotting
Confluent primary cultures of human umbilical vein endothelial
cells were washed twice in HEPES-Tyrode solution and incubated at
37°C with or without various receptor-dependent and -independent
stimuli as described in "Results." Thereafter, cells were washed
with ice-cold HEPES-Tyrode solution containing NaF (100 mmol/L),
Na4P2O7 (15 mmol/L),
Na3VO4 (2 mmol/L), leupeptin (2 µg/mL),
pepstatin A (2 µg/mL), trypsin inhibitor (10 µg/mL), and
phenylmethylsulfonyl fluoride (PMSF, 44 µg/mL) and harvested by
scraping. The cell suspension was centrifuged at 13 000g
for 60 seconds. Cells contained in the pellet were then lysed in buffer
containing 1% (vol/vol) Triton X-100, left on ice for 5 minutes, and
then centrifuged at 10 000g for 10 minutes. Approximately
70 µg protein from the resulting supernatant was separated by either
8% or 12% sodium dodecyl sulfate (SDS)polyacrylamide gel
electrophoresis (PAGE) (Minigel Apparatus, Bio-Rad) and transblotted
onto nitrocellulose paper. After washing for 1 hour at room temperature
in a buffer containing Tris (50 mmol/L, pH 7.5), NaCl (200 mmol/L),
0.2% Triton X-100, 3% bovine serum albumin, and 10% horse serum,
blots were incubated with either a mouse monoclonal
anti-phosphotyrosine antibody (1 µg/mL) (UBI) or a rabbit polyclonal
antiMAP kinase (erk-CT) antibody (1 µg/mL) (UBI) at 4°C for 18
hours in a buffer containing Tris (500 mmol/L, pH 7.5), NaCl (200
mmol/L), and 0.2% Triton X-100. Blots were subsequently washed and
incubated with a peroxidase-conjugated anti-mouse or anti-rabbit
antibody (Amersham) at room temperature for 1 hour, washed again, and
stained by the addition of diaminobenzidine (0.5 mg/mL) and
H2O2 (5 µL of a 30% stock solution) as
previously described.11 Prestained molecular weight marker
proteins (Bio-Rad) and a positive-control protein preparation from A431
cells containing the phosphorylated epidermal growth factor receptor
(UBI) were used as standards for SDS-PAGE and phosphotyrosine
immunodetection, respectively. All of the bands recognized by the
anti-phosphotyrosine antibody could be competitively displaced by
phosphotyrosine (not shown).
Immunoprecipitation of Tyrosine-Phosphorylated Proteins
The Triton-soluble 10 000g supernatant from control
and bradykinin-stimulated confluent primary cultures of human umbilical
vein endothelial cells was prepared as described, and 100 µg protein
was diluted in 500 µL RIPA buffer containing Tris/HCl at pH 7.5 (50
mmol/L), NaCl (150 mmol/L), EGTA (1 mmol/L), sodium desoxycholate
(0.25%), Nonidet P-40 (1%), Na3VO4 (1
mmol/L), NaF (100 mmol/L), Na4P2O7
(15 mmol/L), leupeptin (2 µg/mL), pepstatin A (2 µg/mL), trypsin
inhibitor (10 µg/mL), and PMSF (44 µg/mL). Tyrosine-phosphorylated
proteins were precipitated by shaking gently overnight at 4°C with 30
µL of an anti-phosphotyrosine antibody covalently linked to agarose
(UBI). The agarose was then recovered by centrifugation, washed five
times with RIPA buffer, resuspended in SDSsample buffer, and boiled
for 10 minutes. The resulting supernatant was analyzed by SDS-PAGE and
Western blotting.
Measurement of [Ca2+]i
For the measurement of
[Ca2+]i, endothelial cells were
loaded with the fluorescent Ca2+-sensitive dye fura
2 by incubation with 3 µmol/L fura 2-AM and 0.025% (wt/vol) pluronic
F-127 at 37°C for 90 minutes. Thereafter, the coverslips were washed
in HEPES-modified Tyrode's solution of the following composition
(mmol/L): NaCl 132, KCl 4, CaCl2 1, MgCl2 0.5,
HEPES 9.5, and glucose 5. Coverslips were then mounted in a flow
chamber, superfused with HEPES-Tyrode solution containing 0.3% bovine
serum albumin, and placed on the stage of an inverted microscope
(Diaphot-TMB, Nikon). [Ca2+]i was
determined fluorometrically by using continuous rapid alternating
excitation from dual monochromators set at 340 and 380 nm (Deltascan,
Photon Technology). Incident light passed through a filter block (BA
480, Nikon) and was focused onto the sample by means of an objective
(Fluor 40, Nikon). Emitted light was collected by the objective, and
fluorescence >480 nm was detected by a photon-counting photomultiplier
(model D-104, Photon Technology). Photomultiplier digital output was
collected by an IBM-compatible computer. At the end of each experiment,
cells were superfused with a buffer containing Ca2+
(1.5 mmol/L) and ionomycin (1 µmol/L). After a stable 340/380 ratio
was achieved (maximum, Rmax), the buffer was changed
to one containing EGTA (20 mmol/L) and ionomycin (1 µmol/L; minimum,
Rmin). The background signal was determined after the
addition of MnCl2 (10 mmol/L) to the perfusate, and
[Ca2+]i was calculated by assuming a
dissociation constant for fura 2 at 37°C of 220 nmol/L and using the
following relation as described by Grynkiewicz et
al12 :
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Statistical Analysis
Unless otherwise indicated, data are expressed as
mean±SEM. Statistical evaluation was performed by using Student's
t test for unpaired data, one-way ANOVA followed by a
Bonferroni t test, or ANOVA for repeated measures, where
appropriate. Values of P<.05 were considered statistically
significant.
| Results |
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Stimulation of endothelial cells with bradykinin (100 nmol/L) resulted
in the time-dependent phosphorylation of two low molecular weight
proteins (Fig 1
). The smaller of the two proteins (
42 kD) could be
detected in unstimulated cells, but in bradykinin-stimulated cells no
increase in tyrosine phosphorylation was evident after 30 seconds or 1
minute (not shown). However, a significant increase in tyrosine
phosphorylation was detected 2 minutes after the addition of
bradykinin. The slightly larger protein (
44 kD), although not
apparent in control cells, was clearly tyrosine-phosphorylated 5
minutes after the addition of bradykinin. Maximal phosphorylation of
both proteins was detected after 5 minutes, was decreased slightly by
10 minutes, but was still detectable as long as the cells remained in
contact with the agonist. Bradykinin-stimulated tyrosine
phosphorylation was found to be reversible, since a time-dependent
decrease of both bands was observed after agonist removal, with no
phosphorylation being detected after 10 minutes (not shown).
Stimulation of endothelial cells with histamine (1 µmol/L, 5
minutes) and thapsigargin (30 nmol/L, 15 minutes) resulted in the
tyrosine phosphorylation of two low molecular weight proteins (
42-
and 44-kD), which were identical to those phosphorylated after
stimulation with bradykinin (Fig 2
). Similar results
were also obtained with ionomycin (0.5 µmol/L, 5 minutes; not
shown).
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Effect of Ca2+ Removal on Bradykinin-Stimulated
Tyrosine Phosphorylation
Protein tyrosine phosphorylation of solvent-treated endothelial
cells was unaltered by incubation for up to 15 minutes in
Ca2+-free HEPES-Tyrode solution containing EGTA (0.2
mmol/L) (Fig 3
). The bradykinin-stimulated tyrosine
phosphorylation of the 42- and 44-kD proteins, on the other hand, was
found to be transient and was markedly attenuated by the removal of
extracellular Ca2+. Indeed the 42- and the 44-kD
proteins could only barely be detected 5 minutes after the addition of
bradykinin (Fig 3
).
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Effect of Genistein and Piceatannol on Bradykinin-Stimulated
Tyrosine Phosphorylation
Preincubation of endothelial cells with the tyrosine kinase
inhibitor genistein (100 µmol/L, 15 minutes) was without effect on
protein tyrosine phosphorylation in unstimulated cells (Fig 4
). However, addition of bradykinin (100 nmol/L, 5
minutes) to genistein-treated cells did not result in tyrosine
phosphorylation of either the 42- or 44-kD proteins. Similarly,
piceatannol (10 µmol/L), a tyrosine kinase inhibitor that acts via a
mechanism distinct from that of genistein, also prevented the
bradykinin-stimulated tyrosine phosphorylation of the two proteins.
However, incubation with piceatannol alone resulted in the appearance
of at least two proteins of between 53 and 57 kD (Fig 4
).
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Effect of Bradykinin on MAP Kinase Phosphorylation in Human
Endothelial Cells
Immunoblotting of protein from confluent primary cultures of human
endothelial cells using a polyclonal antiMAP kinase antibody revealed
the presence of two MAP kinases in endothelial cells corresponding to
molecular weights of
42 and
44 kD. Within 5 minutes of
stimulation with bradykinin (100 nmol/L), a slight but distinct shift
in the position of approximately one half of the 42-kD MAP kinase
toward a lower mobility could be detected (Fig 5
, left).
This decrease in the mobility of the 42-kD protein presumably reflects
the phosphorylation of the MAP kinase at threonine and tyrosine
residues, which is required for enzyme activity.13
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To substantiate the hypothesis that stimulation of endothelial cells
with bradykinin results in the tyrosine phosphorylation of the 42- and
44-kD MAP kinases, tyrosine-phosphorylated proteins present in the
cell extract were immunoprecipitated by using an anti-phosphotyrosine
antibody. When immunoprecipitated protein from control cells was
blotted with antiMAP kinase antibody, only one band (
44 kD) was
visible, an observation that is consistent with a basal level of
tyrosine phosphorylation under control conditions (Fig 5
, right). After
cell stimulation with bradykinin, the density of the 44-kD band
increased at least fourfold, and a less densely stained band of
42
kD became clearly visible. Although the 44-kD MAP kinase was more
readily immunoprecipitated than the 42-kD isoform, these results
indicate that bradykinin does indeed stimulate the tyrosine
phosphorylation of both the 42- and 44-kD MAP kinases.
Effect of Tyrosine Kinase Inhibition on Bradykinin-Stimulated
Increases in [Ca2+]i
The effect of tyrosine kinase inhibition on
Ca2+ signaling was investigated in endothelial cells
loaded with the Ca2+-sensitive dye fura 2.
Superfusion of endothelial cells with bradykinin (30 nmol/L) resulted
in a rapid and transient peak increase in
[Ca2+]i followed by a maintained
elevation of [Ca2+]i above resting
values (Fig 6A
). These observations are in accordance
with previously reported data.1 2 14
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The tyrosine kinase inhibitor genistein (100 µmol/L) was without
effect on basal levels of
[Ca2+]i, with resting
[Ca2+]i being 87±15 nmol/L in control
cells and 82±34 nmol/L in genistein-treated cells (P=NS,
n=5). However, genistein (100 µmol/L) attenuated the peak increase in
[Ca2+]i after the addition of
bradykinin (inhibition was 54±6%; P<.01, n=5) as well as
the plateau phase of the Ca2+ response (inhibition
was 62±3%; P<.01, n=5) as measured 12 minutes after the
addition of bradykinin (Fig 6A
).
In the absence of extracellular Ca2+, the
bradykinin-stimulated increase in
[Ca2+]i was similar in solvent- and
genistein-treated cells ([Ca2+]i being
265.8±49.9 nmol/L and 262±15 nmol/L, respectively; P=NS,
n=5) (Fig 6B
). Addition of extracellular Ca2+ (1
mmol/L) to these cells caused a characteristic rapid increase in
[Ca2+]i, which was
significantly attenuated in genistein-treated cells. After the
inhibition of tyrosine kinases, the peak increase in
[Ca2+]i was inhibited by 71.3±4.6%
(P<.001, n=5), and the plateau level of
[Ca2+]i 6 minutes after the addition
of extracellular Ca2+ was attenuated by 60.6±12%
(P<.05, n=5) (Fig 6B
).
Pretreatment of endothelial cells with the tyrosine kinase inhibitor
piceatannol (10 µmol/L) resulted in qualitatively the same effects as
genistein on the bradykinin-stimulated Ca2+ response
(Fig 6
). Although the initial Ca2+ increase in
response to bradykinin tended to be decreased in piceatannol-treated
cells, this effect failed to attain statistical significance.
Effect of Tyrosine Kinase Inhibition on Thapsigargin-Stimulated
Increases in [Ca2+]i
Thapsigargin (30 nmol/L), which (independent of an increase in
inositol phosphates) increases [Ca2+]i
by inhibiting the microsomal
Ca2+-ATPase,15 16 induced a slightly
delayed but long-lasting increase in endothelial
[Ca2+]i (Fig 7A
). This
response was significantly attenuated in genistein-treated cells, with
[Ca2+]i being 450±25 nmol/L in
solvent-pretreated cells compared with 161±28 nmol/L in
genistein-pretreated cells (P<.0001, n=5), as observed 12
minutes after the addition of thapsigargin (Fig 7A
).
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In the absence of extracellular Ca2+, the
thapsigargin-stimulated increase in
[Ca2+]i was similar in solvent- and
genistein-treated cells, with [Ca2+]i
being 81±12 and 80±4 nmol/L, respectively (P=NS, n=5) (Fig 7B
). Addition of extracellular Ca2+ (1 mmol/L)
caused a rapid increase in
[Ca2+]i, which was
significantly attenuated by the inhibition of tyrosine kinases. In
genistein-treated cells, the increase in
[Ca2+]i observed 8 minutes after the
addition of extracellular Ca2+ was only 30.0±2.0%
(P<.0001, n=5) of that detected in solvent-treated cells
(Fig 7B
).
Pretreatment of endothelial cells with the tyrosine kinase inhibitor
piceatannol (10 mmol/L) also significantly attenuated the portion of
the Ca2+ response to thapsigargin that is normally
attributed to Ca2+ influx (Fig 7
).
| Discussion |
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The occurrence of phosphorylated tyrosine as a stable yet reversible
modification of proteins in endothelial cells implies the existence of
one or more protein tyrosine kinases and also of protein tyrosine
phosphatases. The last 5 years has seen a vast increase in the number
of systems in which tyrosine phosphorylation has been implicated (for
review see Reference 1717 ). Similarly, the list of known
tyrosine-phosphorylated substrate proteins has also expanded and now
includes many proteins with known enzymatic activities or assumed
functions, such as the
1 and
2 isoforms of phospholipase C (PLC),
phosphatidylinositol 3-kinase, MAP kinase, the Ras GTPaseactivating
protein, and the putative nucleotide exchange factor
Vav.18 Although endothelial cells have been reported to
contain a modest level of tyrosine kinase activity,19
their involvement in the intracellular signal transduction cascade was
thought to be restricted mainly to events instigated after the
occupancy of growth factor receptors known to have inherent tyrosine
kinase activity.19 This concept would now appear to be an
underestimation of the role of tyrosine phosphorylation in cell
signaling. Indeed, there is an increasing amount of evidence suggesting
that after occupancy, G proteincoupled receptors, which are devoid of
inherent protein tyrosine kinase activity, can in fact rapidly induce
tyrosine phosphorylation of cellular substrate proteins and of the
receptors themselves. For example, the mitogenic actions of endothelin
involve a protein tyrosine kinasebased mechanism,20 and
angiotensin II and vasopressin are able to activate tyrosine kinase
pathways to cause contraction in certain intact smooth muscle
preparations.21 More recently, a fusion protein comprising
the intracellular tail of the AT1 receptor was found to be
an excellent substrate for the src family of protein
tyrosine kinases.22 This phenomenon, at least in the case
of the AT1 receptor, has been shown to be linked to the
tyrosine phosphorylation of PLC-
1 and to a subsequent increase in
the formation of IP3.23 Since G
proteincoupled receptors share a similar molecular architecture, this
situation could also apply to the kinin and histamine receptors.
Indeed, the C-terminal tail of the B2-kinin receptor, which
contains tyrosine residues susceptible to modification by
phosphorylation,24 25 has recently been reported to be
tyrosine-phosphorylated after occupancy.26 The recruitment
of a nonreceptor tyrosine kinase into the signal transduction pathway
for a variety of agonists that act via receptors lacking intrinsic
tyrosine kinase activity would therefore appear to be a more general
phenomenon.
However, tyrosine phosphorylation of the kinin or histamine receptor in human endothelial cells is unlikely to be able to account for the effects observed in the present study, since the same two cytosolic proteins were also tyrosine-phosphorylated after cell stimulation with the receptor-independent agonist ionomycin and the Ca2+-ATPase inhibitor thapsigargin, both of which enhance Ca2+ influx by stimulating the store-regulated Ca2+ influx pathway.27 28 From the experiments using an antibody that recognizes the 43-, 42-, and 44-kD MAP kinases, encoded by the erk1, mapk, and mpk genes, we were able to show that the 42-/44-kD doublet corresponds exactly to the MAP kinases present in endothelial cells. Moreover, phosphotyrosine immunoprecipitation experiments revealed that bradykinin stimulation is indeed associated with an increased tyrosine phosphorylation of the 42- and 44-kD MAP kinases when compared with solvent-treated cells.
The tyrosine phosphorylation and activation of MAP kinase is, on the other hand, unlikely to be involved in the very early steps of signal transduction following cell activation with bradykinin, histamine, and thapsigargin. Rather, because of the dependence of tyrosine phosphorylation of the 42- and 44-kD proteins on extracellular Ca2+, it would appear that their activation occurs at a point downstream to the increase in [Ca2+]i. This would fit with the observations that no increase in MAP kinase tyrosine phosphorylation was observed until 2 minutes after the addition of bradykinin and that maximum tyrosine phosphorylation was relatively delayed, being observed 5 minutes after cell stimulation. However, the increase in [Ca2+]i was relatively rapid, with the peak increase being recorded 1 minute after bradykinin administration. Although our finding that an increase in [Ca2+]i could indeed be sufficient to activate MAP kinase in endothelial cells was at first sight unexpected, it is supported by the observation that both the Ca2+ ionophore and thapsigargin have been shown to stimulate the 44- and 42-kD MAP kinase isoenzymes in human fibroblasts and epidermal carcinoma cells.29 In the latter cell types, however, extracellular Ca2+ was not required to activate the MAP kinases, thus suggesting that activation required only the release of intracellular Ca2+. However, the Ca2+ dependency of MAP kinase activation is not a generally observed phenomenon, since an increase in [Ca2+]i failed to activate MAP kinase in COS cells.30
The most unexpected finding of the present study was the
interdependence of Ca2+ and tyrosine
phosphorylation. On one hand, an increase in
[Ca2+]i was essential for maintained
tyrosine phosphorylation; on the other hand, the
Ca2+ influx was modulated by tyrosine kinase
inhibitors. In fura 2loaded endothelial cells, the tyrosine kinase
inhibitors genistein and piceatannol, which prevented the
agonist-induced tyrosine phosphorylation, attenuated the
Ca2+ response to bradykinin and thapsigargin. In the
presence of extracellular Ca2+, both the peak
and plateau phases of the Ca2+ response to these
agonists were altered. However, the experiments performed in the
absence of Ca2+ demonstrated that inhibition of
tyrosine kinase activity selectively attenuated the plateau phase of
the increase in [Ca2+]i by
55%.
The phenomenon of Ca2+ overshoot, which is observed
after the addition of Ca2+ to cells stimulated in
the absence of extracellular Ca2+, clearly
shows that depletion of intracellular stores by agonists results in an
increased permeability of the plasma membrane to
Ca2+ and is consistent with previously published
data.4 5 Since inhibition of tyrosine kinases specifically
attenuates this portion of the Ca2+ response
following stimulation of endothelial cells with either
receptor-dependent or -independent agonists, the effect of tyrosine
kinase inhibition appeared to be mostly associated with the influx of
Ca2+ from the extracellular space. These findings
suggest that not only are changes in
[Ca2+]i able to alter protein tyrosine
phosphorylation but that tyrosine phosphorylation of specific proteins
may be involved in the regulation of
[Ca2+]i. Since increases in
Ca2+ lead to tyrosine phosphorylation and tyrosine
phosphorylation leads to increases in
[Ca2+]i, it could be expected
that a positive feedback would be rapidly established, resulting in a
cellular Ca2+ overload. However, the level of
phosphorylation of a protein reflects the ratio of its rates of
phosphorylation and dephosphorylation, which are determined by the
intrinsic activities of the kinase(s) and phosphatase(s) for which the
protein is a substrate. Therefore, it is more than likely that a
positive feedback on Ca2+ influx in endothelial
cells is avoided by the concomitant bradykinin-induced activation of
protein tyrosine phosphatase(s). Evidence to support the existence of
such a control mechanism can be derived from experiments using a number
of cell types, including cultured human endothelial cells (I. Fleming,
unpublished observations), in which protein tyrosine phosphatase
inhibitors have been found to instigate an increase in
[Ca2+]i.31 32
Recently, there have been several reports linking agonist-induced Ca2+ mobilization with activation of tyrosine kinases in platelets,6 liver epithelial cells,9 and human foreskin fibroblasts.33 In platelets, for example, tyrosine phosphorylation of a group of proteins was reported to be transiently elevated after a thrombin-stimulated increase in [Ca2+]i,6 a phenomenon later shown to be closely correlated with the synthesis of phosphatidylinositol 3,4-bisphosphate.7 Thrombin-induced protein tyrosine phosphorylation was sensitive to the chelation of intracellular Ca2+ and could be mimicked by inhibiting the Ca2+-ATPase, either reversibly by decreasing temperature or irreversibly by the administration of thapsigargin.6 These observations hinted that, at least in platelets, the mobilization of intracellular Ca2+ stores favors tyrosine phosphorylation, whereas homeostatic levels of Ca2+ in storage compartments favor tyrosine dephosphorylation of specific proteins.6 In addition, protein tyrosine kinase inhibitors such as genistein have been reported to inhibit the thrombin-induced increase in platelet [Ca2+]i and the subsequent aggregation.8 However, the involvement of tyrosine kinases in Ca2+ signaling is not necessarily a universal phenomenon, since tyrosine kinase activity is not required for the activation of Ca2+ influx pathways in all cell types.34 35 36
In the present study, the two compounds that were used to
inhibit protein tyrosine kinase activity have distinctly different
mechanisms of action. While genistein inhibits protein tyrosine kinases
by competing with ATP rather than the substrate, piceatannol
specifically inhibits a number of tyrosine kinases, including p56lck,
p60src, and the epidermal growth factor receptor by competing with the
peptide or protein substrate binding site.37 In
unstimulated endothelial cells, genistein was without effect on the
constitutively tyrosine-phosphorylated proteins, whereas incubation of
the cells with piceatannol led to the detection of a band of proteins
of
55 to 57 kD. This band is unlikely to represent a
protein(s) that is activated by the compound per se but most probably
represents an increase in the mobility of a larger cytosolic
protein, as a consequence of its tyrosine dephosphorylation during the
2-hour incubation period with the tyrosine kinase inhibitor. There have
been a number of concerns expressed regarding the specificity of
genistein, which at concentrations >100 µmol/L, has been shown to
inhibit protein kinase A. However, since the catalytic subunit of
protein kinase A is apparently not affected by treatment with
piceatannol37 and since genistein and piceatannol produced
essentially the same effects, it would appear that the effect of these
compounds on agonist-stimulated Ca2+ influx is a
consequence of their shared capacity to inhibit protein tyrosine kinase
activity.
Comparison of the kinetics of agonist-induced increase in [Ca2+]i with that of tyrosine phosphorylation suggests that the tyrosine-phosphorylated protein involved in regulating Ca2+ influx is in all probability not a MAP kinase. Rather, a more likely candidate would be a protein either attached to the membrane or whose presence is masked by the constitutively tyrosine-phosphorylated proteins detected in the cytosol of control cells. Moreover, the protein involved in regulating Ca2+ influx would be expected to be tyrosine-phosphorylated immediately after addition of the agonist, even in the absence of extracellular Ca2+, and to remain phosphorylated until store filling was complete. Such a tyrosine-phosphorylated protein has been described in platelets6 and has been tentatively identified as the microfilament-associated protein vinculin.38 To date, however, there has been no description of such a tyrosine-phosphorylated protein in endothelial cells.
It is likely that the activation of tyrosine kinases represents
only one link in the chain of events involved in regulating
Ca2+ influx into endothelial cells. Indeed, a number
of recent reports have suggested that the regulation of
Ca2+ influx also involves serine-threonine
kinases39 and small G proteins.40 41 The
involvement of such a kinase cascade in endothelial cells is suggested
by our observation that okadaic acid and calyculin A, two potent
serine-threonine phosphatase inhibitors,42 attenuated
Ca2+ influx into human umbilical vein endothelial
cells (I. Fleming, unpublished observation). Therefore, it would seem
that the intracellular signal transmitted by empty
Ca2+ stores to initiate a transmembranous
Ca2+ influx involves a process in which the
dephosphorylation of a serine-threoninephosphorylated protein and the
activation of a protein tyrosine kinase are both implicated. Although
the kinase cascade involved has not been identified, there is a certain
amount of circumstantial evidence to support the hypothesis that
activation of the Ras-GTP/Raf pathway, which forms part of the kinase
cascade upstream from the MAP kinase, is important in
Ca2+ influx. For example, inhibition of GTP
hydrolysis by introducing nonhydrolyzable guanine nucleotide analogues,
such as GTP
S,1 into cells not only inhibits Ras but
also inhibits thapsigargin-induced Ca2+ entry into
mouse lacrimal acinar cells40 and rat basophilic leukemia
cells.41 In addition, it has been shown that GTP increased
the size of the IP3-sensitive Ca2+ store
in digitonin-permeabilized hepatocytes.43
In summary, we propose that the activation of tyrosine kinases forms part of the cascade of events linking the emptying of internal Ca2+ stores with the activation of the Ca2+ influx pathway in human endothelial cells. One hypothesis that could account for such findings would be that Ca2+ released from intracellular stores might activate a Ca2+-dependent tyrosine kinase(s), which could then in turn activate a Ca2+ influx pathway. In addition, our finding that the MAP kinase was tyrosine-phosphorylated, and presumably activated, in endothelial cells after an increase in [Ca2+]i has wide-ranging implications for these cells, since MAP kinase recognizes many different substrates in the cell, including growth factor receptors, microtubule-associated proteins, specific serine-threonine protein kinases, phospholipase A2, and transcription factors.44 Although the involvement of the Ras-GTP/Raf/MAP kinase pathway in Ca2+ signaling is at the moment speculative, its activation after increases in endothelial [Ca2+]i, such as that seen in response to shear stress,45 46 may open up a whole new field of research in endothelial cell physiology as a means of linking this mechanical signal to alterations in gene transcription.
| Acknowledgments |
|---|
Received July 28, 1994; accepted December 13, 1994.
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S Greco, C Storelli, and S Marsigliante Protein kinase C (PKC)-{delta}/-{varepsilon} mediate the PKC/Akt-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 in MCF-7 cells stimulated by bradykinin J. Endocrinol., January 1, 2006; 188(1): 79 - 89. [Abstract] [Full Text] [PDF] |
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A. Hus-Citharel, X. Iturrioz, P. Corvol, J. Marchetti, and C. Llorens-Cortes Tyrosine Kinase and Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase Differentially Regulate Intracellular Calcium Concentration Responses to Angiotensin II/III and Bradykinin in Rat Cortical Thick Ascending Limb Endocrinology, January 1, 2006; 147(1): 451 - 463. [Abstract] [Full Text] [PDF] |
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A. A. S. Aromolaran and L. A. Blatter Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells Am J Physiol Cell Physiol, December 1, 2005; 289(6): C1426 - C1436. [Abstract] [Full Text] [PDF] |
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L. M. F. Leeb-Lundberg, F. Marceau, W. Muller-Esterl, D. J. Pettibone, and B. L. Zuraw International Union of Pharmacology. XLV. Classification of the Kinin Receptor Family: from Molecular Mechanisms to Pathophysiological Consequences Pharmacol. Rev., March 1, 2005; 57(1): 27 - 77. [Abstract] [Full Text] [PDF] |
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M. H Wu, S. Y Yuan, and H. J Granger The protein kinase MEK1/2 mediate vascular endothelial growth factor- and histamine-induced hyperpermeability in porcine coronary venules J. Physiol., February 15, 2005; 563(1): 95 - 104. [Abstract] [Full Text] [PDF] |
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K. Kappert, K. G. Peters, F. D. Bohmer, and A. Ostman Tyrosine phosphatases in vessel wall signaling Cardiovasc Res, February 15, 2005; 65(3): 587 - 598. [Abstract] [Full Text] [PDF] |
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R. Popp, R. P. Brandes, G. Ott, R. Busse, and I. Fleming Dynamic Modulation of Interendothelial Gap Junctional Communication by 11,12-Epoxyeicosatrienoic Acid Circ. Res., April 19, 2002; 90(7): 800 - 806. [Abstract] [Full Text] [PDF] |
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H.-L. Xu, D. L. Feinstein, R. A. Santizo, H. M. Koenig, and D. A. Pelligrino Agonist-specific differences in mechanisms mediating eNOS-dependent pial arteriolar dilation in rats Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H237 - H243. [Abstract] [Full Text] [PDF] |
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