(Received for publication, June 29, 1995; and in revised form, January 12, 1996)
From the
The signal transduction cascade which initiates transmembraneous
influx of Ca into endothelial cells in response to
the discharge of intracellular Ca
stores is thought
to involve a step sensitive to tyrosine kinase inhibition. We
investigated the interrelationship between Ca
signaling and protein tyrosine phosphorylation following cell
stimulation with either the receptor-dependent agonist, bradykinin, or
the protein-tyrosine phosphatase inhibitor, phenylarsine oxide. In
cultured human endothelial cells phenylarsine oxide instigated a
concentration-dependent increase in the intracellular concentration of
free Ca
([Ca
]
). This
increase in [Ca
]
was
not associated with the tyrosine phosphorylation of phospholipase
C
, enhanced formation of inositol 1,4,5-trisphosphate, or the
rapid depletion of intracellularly stored Ca
but was
coincident with the enhanced and prolonged tyrosine phosphorylation of
a number of cytoskeletal proteins. In bradykinin-stimulated cells the
tyrosine phosphorylation of the same cytoskeletal proteins (most
notably 85- and 100-kDa proteins) was transient when cells were
stimulated in the presence of extracellular Ca
, was
maintained under Ca
-free conditions, and was reversed
following readdition of extracellular Ca
. These data
suggest that the tyrosine phosphorylation of 2 cytoskeletal proteins is
determined by the level of Ca
present in
intracellular stores thus indicating a critical role for tyrosine
phosphorylation in the control of capacitative Ca
entry in endothelial cells.
There is a certain amount of evidence to suggest that the
tyrosine phosphorylation of, as yet unidentified, cellular proteins may
be involved in the control of store-regulated or
``capacitative'' Ca entry following the
agonist-induced depletion of intracellular stores in non-excitable
cells. The first evidence for such a role of tyrosine kinases in
intracellular Ca
signaling was obtained in platelets,
in which the tyrosine phosphorylation of a group of proteins was found
to be transiently elevated following stimulation with
thrombin(1, 2, 3) . This thrombin-induced
protein tyrosine phosphorylation could be mimicked by the depletion of
intracellular Ca
stores following inhibition of the
Ca
-ATPase and was sensitive to the chelation of
intracellular Ca
. Repletion of Ca
stores was, on the other hand, associated with a return to basal
phosphorylation levels(1) . These observations inferred that
the depletion of intracellular Ca
stores favors
tyrosine phosphorylation whereas store refilling and the restoration of
homeostatic levels of [Ca
]
favors tyrosine dephosphorylation of specific
proteins(1) . In support of these findings, protein-tyrosine
kinase inhibitors such as genistein were reported to inhibit both the
thrombin-induced increase in [Ca
]
as well as the subsequent aggregation (2) . Since
genistein has been found to attenuate Ca
influx
following cell stimulation with both receptor-dependent and
-independent agonists it would appear that the tyrosine kinase
substrate protein is likely to be intimately involved in the regulation
of Ca
entry processes rather than being linked to
specific cell receptors. Recently we have demonstrated that the
bradykinin as well as the thapsigargin-induced Ca
influx in endothelial cells is also mediated by a tyrosine kinase
inhibitor-sensitive mechanism(4) . Although the chain of events
which result in the enhanced membrane permeability to Ca
remain largely unexplained, these observations suggest that a
tyrosine-phosphorylated protein might be involved in the regulation of
Ca
influx. Therefore the aim of the present study was
to address the hypothesis that protein tyrosine phosphorylation
controls capacitative Ca
influx in human endothelial
cells. To this end we investigated the effects of agonist stimulation
on tyrosine phosphorylation as well as the effects on
[Ca
]
of altering the
balance between tyrosine kinase/phosphatase activity.
Figure 1:
Original tracings illustrating the
concentration-dependent effect of phenylarsine oxide on the
intracellular concentration of free Ca
([Ca
]
) in fura 2-loaded human
endothelial cells. Phenylarsine oxide (1-10 µM) was
added to cells incubated in the absence and presence of the tyrosine
kinase inhibitor genistein (100 µM). Data are
representative of experiments performed using 4-6 different cell
batches.
The tyrosine kinase
inhibitor genistein (100 µM), which has previously been
shown to attenuate bradykinin-stimulated Ca-influx in
endothelial cells(4) , markedly reduced the Ca
response to phenylarsine oxide.
[Ca
]
, as measured 10 min after
the addition of phenylarsine oxide (10 µM), was 522
± 30 nM in solvent-treated cells compared with 197
± 14 nM in cells pretreated with genistein (n = 4, p < 0.01, Fig. 1B).
The
phenylarsine oxide-induced increase in
[Ca]
was completely reversed by
the dithiol reagent 2,3-dimercaptopropanol (50 µM) which
binds to vicinal sulfhydryl groups (Fig. 2). In these
experiments, the subsequent addition of bradykinin (100 nM)
resulted in a normal Ca
response suggesting that the
effects of phenylarsine oxide were completely reversible and that the
inhibitor did not lead to the permanent uncoupling of the
agonist-induced signal transduction pathway.
Figure 2:
The
dithiol agent 2,3-dimercaptopropanol (DMP) reverses the
increase in intracellular Ca ([Ca
]
) elicited
by phenylarsine oxide (PAO). The effects of DMP (50
µM) were tested on fura 2-loaded human endothelial cells
exposed to PAO (10 µM) for 10 min. Following the return of
[Ca
]
to baseline
levels cells were stimulated with the receptor-dependent agonist,
bradykinin (100 nM). The results are presented as the mean
± S.E. of data obtained in five separate
experiments.
In the absence of
extracellular Ca, phenylarsine oxide (10
µM) induced a small, slowly developing, increase in
[Ca
]
(after 10 min
[Ca
]
had increased from 69.7
± 12 nM to 106.5 ± 23 nM, n = 8, p < 0.001; Fig. 3). Subsequent
addition of the Ca
-ATPase inhibitor, thapsigargin
(0.3 µM), resulted in an immediate further increase in
[Ca
]
([Ca
]
increased from 104
± 24 to 218 ± 15 nM; n = 4, p < 0.01) demonstrating that the protein-tyrosine
phosphatase inhibitor did not deplete intracellular Ca
stores. In cells stimulated with phenylarsine oxide in the
absence of extracellular Ca
, the readdition of
Ca
was associated with an immediate increase in
[Ca
]
(Fig. 3).
Figure 3:
Effect of phenylarsine oxide on the
intracellular concentration of free Ca
([Ca
]
) in fura
2-loaded human endothelial cells. The increase in
[Ca
]
in response to
phenylarsine oxide (10 µM) was assayed in
Ca
-free buffer with the subsequent addition of
Ca
(1.5 mM). The results are presented as
the mean ± S.E. of data obtained in eight separate
experiments.
When tyrosine-phosphorylated
proteins were immunoprecipitated from endothelial cells treated with
phenylarsine oxide (10 µM) and blotted with
anti-PLC, a clear signal was apparent in control cells
but could not be detected in cells incubated with the tyrosine
phosphatase inhibitor for up to 5 min (Fig. 4A).
Stimulation of endothelial cells with bradykinin (100 nM), on
the other hand, resulted in a rapid increase in the tyrosine
phosphorylation of PLC
with a 3.5-fold increase in
tyrosine-phosphorylated protein being detected within 30 s (Fig. 4B). The bradykinin-induced tyrosine
phosphorylation of PLC
was relatively transient and
tyrosine phosphorylation of the PLC
returned to near
basal levels within 2 min.
Figure 4:
Phenylarsine oxide fails to enhance the
tyrosine phosphorylation of PLC or to stimulate the
production of inositol 1,4,5-trisphosphate (IP
) in human
endothelial cells. Tyrosine phosphorylated proteins were
immunoprecipitated from cells incubated with either (A)
phenylarsine oxide (10 µM) or (B) bradykinin (100
nM) for the times indicated. PLC
in
immunoprecipitates was detected using a specific antibody as described
under ``Experimental Procedures.'' The results presented are
representative of experiments performed using three different cell
batches. C, inositol 1,4,5-trisphosphate (IP
) was
assessed in cells incubated with either phenylarsine oxide (10
µM;
) or bradykinin (100 nM;
) for the
times indicated. The results are presented as the mean ± S.E. of
data obtained in four separate experiments.
In accordance with its effect on the
tyrosine phosphorylation of PLC, phenylarsine oxide
(10 µM) failed to precipitate an increase in intracellular
levels of IP
at any of the time points measured. In
bradykinin-treated endothelial cells IP
levels increased
7-fold within 10 s and had returned to baseline values within 1 min (Fig. 4C).
These observations demonstrate that the
phenylarsine oxide-induced increase in
[Ca]
is not due to the rapid
depletion of intracellular stores and that activation of a
transmembraneous influx accounts for most of the tyrosine phosphatase
inhibitor-induced increase in
[Ca
]
. As a consequence of its
effects on fura-2 fluorescence it was not possible to repeat these
experiments using a second widely used tyrosine phosphatase inhibitor,
sodium orthovanadate.
Figure 5:
Phenylarsine oxide enhances the tyrosine
phosphorylation of Triton X-100-soluble proteins, including the p42 and
p44 MAP kinases, in human endothelial cells. Triton-soluble proteins
from human endothelial cells were (A) incubated in the
presence or absence of phenylarsine oxide (PAO; 10 µM) for
the indicated times or (B) incubated with PAO (10
µM, 2 min) in the presence or absence of extracellular
Ca, following pretreatment or not with the
intracellular Ca
chelator BAPTA (10 µM;
30 min). Triton X-100-soluble proteins were separated by SDS-PAGE and
tyrosine-phosphorylated proteins were detected using a specific
antiphosphotyrosine antibody as described under ``Experimental
Procedures.'' The results presented are representative of
experiments performed using seven different cell
batches.
In order to evaluate the role of
[Ca]
in phenylarsine
oxide-stimulated tyrosine phosphorylation, cells were either incubated
in a nominally Ca
-free buffer or pretreated with the
intracellular Ca
chelator BAPTA (10 µM,
30 min). Phenylarsine oxide-induced tyrosine phosphorylation of the
42/44-kDa doublet was largely Ca
-dependent since a
slight increase in tyrosine phosphorylation was observed in cells
stimulated in the absence of Ca
but no increase was
observed in BAPTA-treated cells. The tyrosine phosphorylation of the
80-kDa triplet was, on the other hand, largely
Ca
-independent (Fig. 5B).
A second
protein-tyrosine phosphatase inhibitor, sodium orthovanadate (0.3
mM), also resulted in a genistein-sensitive, time-dependent
increase in the tyrosine phosphorylation of the 42- and 44-kDa isoforms
of MAP kinase as well as the 80-kDa triplet (Fig. 6).
Figure 6: Orthovanadate time dependently enhances the tyrosine phosphorylation of Triton X-100-soluble proteins, including the p42 and p44 MAP kinases, in human endothelial cells. Human endothelial cells were incubated in the presence or absence of sodium orthovanadate (0.3 mM) for the times indicated. Proteins were separated by SDS-PAGE and tyrosine-phosphorylated proteins were detected using a specific antiphosphotyrosine antibody as described under ``Experimental Procedures.'' The results presented are representative of experiments performed using two different cell batches.
Figure 7: The phenylarsine oxide-induced increase in the tyrosine phosphorylation of Triton X-100-insoluble (cytoskeletal) proteins is attenuated in the presence of the tyrosine kinase inhibitor genistein and following administration of the dithiol agent 2,3-dimercaptopropanol (DMP). Triton-soluble proteins from human endothelial cells were: A, incubated in the presence or absence of phenylarsine oxide (PAO; 10 µM) for the indicated times; B, incubated with PAO (10 µM, 10 min) in the presence or absence of genistein (100 µM); or C, incubated or not with DMP (50 µM) following exposure to PAO (10 µM) for 10 min. Cytoskeletal proteins were separated by SDS-PAGE and tyrosine-phosphorylated proteins were detected using a specific antiphosphotyrosine antibody as described. The results presented are representative of experiments performed using four different cell batches.
Pretreatment of endothelial cells with the tyrosine kinase inhibitor genistein tended to attenuate the phenylarsine oxide-induced tyrosine phosphorylation although the effects were by no means prevented by the inhibitor (Fig. 7B). Almost complete reversal of the phenylarsine oxide-induced increase in tyrosine phosphorylation was achieved by subsequent addition of 2,3-dimercaptopropanol (50 µM; Fig. 7C).
The removal of
extracellular Ca or the chelation of intracellular
Ca
failed to alter the phenylarsine oxide induced
increase in tyrosine phosphorylation of cytoskeletal proteins (Fig. 8).
Figure 8:
The
phenylarsine oxide (PAO)-induced tyrosine phosphorylation of
cytoskeletal proteins is insensitive to the chelation of either
extracellular or intracellular Ca. Phenylarsine oxide
(10 µM) was added to endothelial cells were pretreated or
not with the intracellular Ca
-chelating compound
BAPTA (10 µM; 30 min) and in the presence or absence of
extracellular Ca
. The tyrosine phosphorylation of
cytoskeletal proteins was detected as described under
``Experimental Procedures.'' The results presented are
representative of experiments performed using three different cell
batches.
In the presence of extracellular
Ca, bradykinin (100 nM) induced an immediate
increase in the tyrosine phosphorylation of 3 Triton-soluble proteins
(60, 77, and 86 kDa). Phosphorylation of these proteins was maximal
after 30 s and returned to control levels over 5 min (Fig. 9A). In the same cells bradykinin also induced
tyrosine phosphorylation of the 42/44-kDa MAP kinase doublet which was
detectable 2 min after agonist stimulation and was maximal after 5 min,
as described previously(4) . Stimulation of endothelial cells
in the absence of extracellular Ca
failed to alter
phosphorylation of the 60-, 77-, or 86-kDa proteins but resulted in
only a transient increase in the tyrosine phosphorylation of the MAP
kinases which was no longer apparent after 5 min (Fig. 9B). Phosphorylation of the 42/44-kDa doublet
reappeared 1 to 2 min after the readdition of extracellular
Ca
to these cells (Fig. 9C).
Pretreatment of endothelial cells with BAPTA abrogated the
bradykinin-induced tyrosine phosphorylation of the p42 and p44 (data
not shown).
Figure 9:
Bradykinin induces the tyrosine
phosphorylation of a series of Triton-soluble endothelial proteins
which demonstrate differing time courses of phosphorylation as well as
differing sensitivity to the presence of extracellular
Ca. Human endothelial cells were incubated in the
presence or absence of bradykinin (100 nM) for the times
indicated. A, cells were stimulated in the presence of
extracellular Ca
and B, in the absence of
extracellular Ca
. C, cells were stimulated
with bradykinin (100 nM; 5 min) in the absence of
extracellular Ca
, thereafter Ca
(1.5 mM) was added for the times indicated. The results
presented are representative of experiments performed using seven
different cell batches.
Bradykinin stimulation resulted in an immediate but
transient increase in the phosphorylation of 4 cytoskeletal proteins
with estimated molecular masses of 85, 100, 110, and 125 kDa which
returned to baseline levels after 5-10 min (Fig. 10A). In the absence of extracellular
Ca, bradykinin induced a distinct increase in the
phosphorylation of the 85- and 100-kDa proteins which was immediately
reversed upon readdition of extracellular Ca
(Fig. 10B).
Figure 10:
Bradykinin induces the tyrosine
phosphorylation of cytoskeletal proteins which demonstrate sensitivity
to the filling state of the intracellular Ca store.
Human endothelial cells were incubated in the presence or absence of
bradykinin (100 nM) for the times indicated. Cells were
stimulated in the presence (A) and absence (B) of
extracellular Ca
. Following cell stimulation for 2
min, Ca
(1 mM) was added for the times
indicated. The results presented are representative of experiments
performed using seven different cell
batches.
Thapsigargin (100
nM) induced the tyrosine phosphorylation of 4 proteins in the
Triton-insoluble fraction corresponding to molecular masses of 85, 100,
110, and 125 kDa. However, the tyrosine phosphorylation of these bands
was not transient, as was observed following cell stimulation with
bradykinin, but was maintained for up to 10 min. Removal of
extracellular Ca did not influence the pattern of
tyrosine phosphorylation which emerged following stimulation with
thapsigargin, and the readdition of extracellular Ca
to depleted cells was not associated with a visible change in the
phosphorylation pattern (Fig. 11).
Figure 11:
The Ca-ATPase inhibitor
thapsigargin induces the maintained tyrosine phosphorylation of several
cytoskeletal proteins. Human endothelial cells were incubated with
either solvent or thapsigargin (TG; 100 nM) for 5 min
in the presence and absence of extracellular Ca
.
Following cell stimulation in Ca
-free buffer for 5
min, Ca
(1 mM) was added and the incubation
stopped after a further 2 min (Ca2+ readd.). The results
presented are representative of experiments performed using six
different cell batches.
Over the last few years there have been several reports that
the transmembraneous influx of Ca is selectively
attenuated in a number of cell types following inhibition of tyrosine
kinases(1, 8, 9, 10) , thus
suggesting that cellular levels of tyrosine phosphorylation play a
determinant role in regulating Ca
entry in
non-excitable cells. In the present study the protein-tyrosine
phosphatase inhibitor, phenylarsine oxide, induced a
concentration-dependent increase in
[Ca
]
and elicited the tyrosine
phosphorylation of a number of endothelial proteins with the most
marked effects being apparent in the Triton X-100-insoluble, or
cytoskeletal, fraction. Both the increase in
[Ca
]
and the enhanced tyrosine
phosphorylation were attenuated in cells pretreated with the tyrosine
kinase inhibitor, genistein, supporting the hypothesis that a
tyrosine-phosphorylated protein may be involved in the regulation of
[Ca
]
in endothelial cells.
In principle there are two ways by which an agonist can induce
capacitative Ca entry. The activation of the
transmembraneous Ca
influx pathway could be
attributed to an indirect, i.e. instigation of capacitative
Ca
entry following the mobilization of
intracellularly stored Ca
, or a direct effect, i.e. activation of a Ca
influx regulatory
protein. In contrast to the receptor-dependent agonist, bradykinin,
phenylarsine oxide failed to tyrosine phosphorylate PLC
or
increase cellular levels of IP
. The tyrosine phosphatase
inhibitor therefore appeared unable to mobilize intracellular
Ca
via the classical signaling pathway associated
with agonist-induced activation of endothelial cells. Inhibition of the
Ca
-ATPase is also unable to account for the
phenylarsine oxide-induced Ca
response. In the
absence of extracellular Ca
phenylarsine oxide had no
immediate effect on [Ca
]
but
when incubated with cells for longer periods did induce a slight
elevation. Subsequent addition of thapsigargin to these cells resulted
in an immediate further increase in
[Ca
]
suggesting that the
tyrosine phosphatase inhibitor did not mobilize Ca
from intracellular stores. However, since the readdition of
Ca
to cells stimulated with phenylarsine oxide in the
absence of extracellular Ca
resulted in a marked and
instantaneous increase in [Ca
]
,
it would appear that the tyrosine phosphatase inhibitor directly
activates a Ca
influx pathway which does not appear
to be regulated by the filling state of intracellular Ca
stores.
Our observation that tyrosine phosphatase inhibitors
appear to be able to activate Ca influx pathways
without first mobilizing intracellular stores is supported by similar
findings in T lymphocytes following the administration of phenylarsine
oxide and pervanadate(11, 12) . These data, together
with the results obtained in the present study, suggest that the
effects of the protein-tyrosine phosphatase inhibitors on
[Ca
]
may instead be a direct
consequence of the enhanced tyrosine phosphorylation of a
Ca
influx regulatory protein. This hypothesis is
supported by the report that transfection of T cells with the
constitutively active tyrosine kinase v-Src results in elevated basal
levels of [Ca
]
as well as in
the exaggeration of agonist-stimulated Ca
responses(13) .
Since the inhibition of
protein-tyrosine phosphatases resulted in an increase in
[Ca]
, it would appear likely
that phosphatase activity plays a crucial role in damping
Ca
influx both in unstimulated cells and following
agonist stimulation. Indeed, the observation that protein-tyrosine
phosphatase inhibitors can themselves induce cellular responses implies
that there is a significant basal activity of protein-tyrosine
phosphatases in cultured human endothelial cells. This was confirmed by
determination of tyrosine phosphatase activity in whole cell lysates.
Moreover, the observation that the tyrosine kinase inhibitor genistein
attenuated both the increase in [Ca
]
and tyrosine phosphorylation initiated by phenylarsine oxide
suggests that a certain basal phosphatase activity is required to
counteract phosphorylation by constitutively active protein-tyrosine
kinases. Thus the dynamic balance between tyrosine kinase and
phosphatase activity may play a central role in the maintenance of
homeostatic levels of [Ca
]
in
unstimulated cells.
Based on current knowledge, the hypothetically
ideal Ca influx-regulatory protein in non-excitable
cells should be ``activated'' immediately after
agonist-induced emptying of intracellular Ca
stores,
even in the absence of extracellular Ca
, and to
remain so until store filling is accomplished. Ideally this protein
should be membrane-associated, either permanently or temporarily, and
preferably linked by some manner or means to the cation channel by
which Ca
enters the cell. To identify proteins which
conform with these criteria the tyrosine phosphorylation of proteins
from cells stimulated with bradykinin, thapsigargin, and the tyrosine
phosphatase inhibitors was monitored in Triton X-100-soluble and
-insoluble fractions. In the soluble fraction, bradykinin induced the
rapid and transient phosphorylation of 3 proteins which contrasted with
the relatively slow tyrosine phosphorylation of the 42- and 44-kDa
isoforms of MAP kinase, as described previously(4) . Cell
stimulation in the absence of extracellular Ca
was
without effect on the phosphorylation of the
60-, 77-, and 86-kDa
proteins whereas agonist-induced phosphorylation of the 42- and 44-kDa
isoforms of the MAP kinase was critically dependent on an increase in
[Ca
]
. The
Ca
-ATPase inhibitor, thapsigargin, elicited
essentially the same effects on Triton-soluble proteins. Sodium
orthovanadate and phenylarsine oxide, however, induced the maintained,
Ca
-independent phosphorylation of a protein triplet
of
80 kDa.
In the cytoskeletal fraction bradykinin also evoked
a rapid and transient tyrosine phosphorylation of 4 proteins (85, 100,
110, and 125 kDa) which appeared identical to proteins permanently
tyrosine phosphorylated following application of phenylarsine oxide. In
contrast to the effects seen in the presence of extracellular
Ca, a maintained tyrosine phosphorylation of these
proteins was observed in cells stimulated following the removal of
extracellular Ca
. Readdition of extracellular
Ca
to these Ca
-depleted cells was
associated with the transient dephosphorylation and maintained
rephosphorylation of the 110- and 125-kDa proteins and the sustained
dephosphorylation of the 85- and 100-kDa proteins. In similar
experiments using thapsigargin, the reapplication of extracellular
Ca
to depleted cells did not affect tyrosine
phosphorylation of the 125-kDa protein or result in the
dephosphorylation of the 85- and 100-kDa proteins. This observation
was, however, not unexpected since, in the continued presence of the
Ca
-ATPase inhibitor refilling of intracellular
Ca
stores is antagonized, thus the store remains
empty although the signal for refilling, is sustained. In
bradykinin-stimulated cells, however, store refilling could be
accomplished following Ca
readdition and the tyrosine
phosphorylation of the 85- and 100-kDa proteins was transient. It would
therefore appear that the tyrosine phosphorylation of the 85- and
100-kDa proteins mirrors the filling state of intracellular
Ca
stores, thus these two cytoskeletal proteins fit
the requirements of the hypothetical Ca
influx
regulatory protein. Although similar experiments involving
intracellular Ca
depletion and repletion in platelets
have also demonstrated the existence of tyrosine-phosphorylated
proteins apparently sensitive to the filling state of the
Ca
store (1, 14) , the reported
apparent molecular weights of these proteins differs from that of
proteins displaying similar characteristics in the present study.
In
addition to the putative tyrosine-phosphorylated Ca influx regulatory protein, a number of other mechanisms have been
proposed to regulate capacitative Ca
entry.
Nonhydrolyzable analogues of GTP, such as GTP
S, have been shown to
interfere with Ca
signaling in a number of cell
types. The inhibitory effect of these analogues occurs at some point
after the release of intracellular Ca
and prior to
the activation of Ca
influx. These effects can be
prevented by GTP thus implying that a small G protein is involved in
communicating the empty state of intracellular Ca
stores to the plasma membrane (15, 16, 17, 18) . The role of the
``calcium influx factor,'' a small, phosphate-containing,
non-protein factor termed originally isolated from Jurkat T
lymphocytes(19) , as an exclusive messenger for capacitative
Ca
entry has recently been questioned since the
lymphocyte-derived factor has also been demonstrated to mobilize
Ca
from intracellular stores(20) .
Involvement of the cytochrome P-450 monooxygenase in the regulation of
Ca
influx has also been proposed on the basis of
observations that a number of chemically distinct P-450 inhibitors,
such as the imidazole anti-fungal agents, potently inhibited
Ca
influx in endothelial cells and
platelets(21, 22, 23) . This hypothesis is
supported by the findings that the induction of P-450 by
-naphtoflavone, potentiated agonist-induced Ca
influx and that the P-450 product, 5,6-epoxyeicosatrienoic acid,
activated Ca
entry into endothelial cells without
prior depletion of intracellular Ca
(24) .
Such observations suggest that the regulation of capacitative
Ca
entry into endothelial cells is a complex process
likely to involve the activation of protein tyrosine kinases and
phosphatases, small G proteins, serine/threonine phosphatases, and
probably also the cytochrome P-450 monooxygenase.
In summary, in the
present study we observed that treatment of endothelial cells with
protein-tyrosine phosphatase inhibitors resulted in the prolonged
tyrosine phosphorylation of 2 cytoskeletal proteins and an increased
Ca influx via a mechanism independent of
intracellular Ca
store depletion. Our findings
strongly suggest that the tyrosine phosphorylation of both cytoskeletal
proteins mirrors the filling state of the intracellular Ca
store and that they play a central role in the regulation of
capacitative Ca
entry.