ATP induces dephosphorylation of myosin light chain in
endothelial cells
T.
Noll,
M.
Schäfer,
U.
Schavier-Schmitz, and
H. M.
Piper
Physiologisches Institut, Justus-Liebig-Universität, D-35392
Giessen, Germany
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ABSTRACT |
In cultured porcine aortic endothelial monolayers, the
effect of ATP on myosin light chain (MLC) phosphorylation, which
controls the endothelial contractile machinery, was studied. ATP (10 µM) reduced MLC phosphorylation but increased cytosolic
Ca2+ concentration ([Ca2+]i).
Inhibition of the ATP-evoked [Ca2+]i rise by
xestospongin C (10 µM), an inhibitor of the inositol trisphosphate-dependent Ca2+ release from endoplasmic
reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC
dephosphorylation was prevented in the presence of calyculin A (10 nM),
an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates
MLC dephosphorylation in a Ca2+-independent manner. In the
presence of calyculin A, MLC phosphorylation was incremented after
addition of ATP, an effect that could be abolished when cells
were loaded with the Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
acetoxymethyl ester (10 µM). Thus ATP also activates a
Ca2+-dependent kinase acting on MLC. In summary, ATP
simultaneously stimulates a functional antagonism toward both
phosphorylation and dephosphorylation of MLC in which the
dephosphorylation prevails. In endothelial cells, ATP is the first
physiological mediator identified to activate MLC dephosphorylation by
a Ca2+-independent mechanism.
adenosine 5'-triphosphate; myosin light chain phosphorylation; protein phosphatases
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INTRODUCTION |
ENDOTHELIAL CELLS
POSSESS a contractile apparatus, resembling the one found in
smooth muscle cells (18, 27). They contain the contractile
elements actin and myosin and accompanying regulatory proteins
(18). Phosphorylation of the regulatory myosin light chain
(MLC) leads to activation of the endothelial contractile elements
(26). An important second messenger regulating MLC phosphorylation is cytosolic Ca2+ concentration
([Ca2+]i); this controls the activity of
endothelial Ca2+/calmodulin-dependent MLC kinase (23,
26, 27). It has been shown that mediators like thrombin
(6, 19) and histamine (12, 13) can cause
hyperpermeability of the endothelial barrier by eliciting an increase
in MLC phosphorylation and endothelial cell contraction via a
receptor-mediated transient rise in cytosolic Ca2+
(6, 20). Endothelial cells also express protein
phosphatases PP-1 and PP-2A (3, 10, 24), which can also
influence the phosphorylation state of MLC. We and others showed that
inhibition of these protein phosphatases leads to hyperpermeability of
the endothelial barrier (3, 10, 24). At present, little is known about the functional antagonism between MLC kinase and myosin phosphatases when these are stimulated simultaneously in endothelial cells.
We found recently (16) that the purine receptor agonist
ATP causes a transient rise in [Ca2+]i and
yet a reduction of barrier permeability in endothelial monolayers,
derived from different mammalian species and vascular provinces such as
porcine aorta, porcine pulmonary artery, bovine aorta, and human
umbilical vein. The effects were specific for the nucleotide and could
not be imitated by adenosine. The observations have suggested that, in
endothelial cells, ATP simultaneously activates the antagonistic
mechanisms controlling the phosphorylation state of MLC, i.e., a
Ca2+-dependent protein kinase and a
Ca2+-independent protein phosphatase acting on MLC. The
present study was undertaken to analyze these mechanisms in intact
endothelial cells. The results demonstrate that ATP predominantly
activates dephosphorylation of MLCs. ATP is the first physiological
mediator for which such a Ca2+-independent activation has
been shown in endothelial cells.
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MATERIALS AND METHODS |
Cell cultures.
Endothelial cells from porcine aorta were isolated and cultured as
previously described (25). Confluent cultures of primary endothelial cell were trypsinized in phosphate-buffered saline [composed of (in mM) 137 NaCl, 2.7 KCl, 1.5 KH2PO4, and 8.0 Na2HPO4, at pH 7.4, supplemented with 0.05%
(wt/vol) trypsin, and 0.02% (wt/vol) EDTA] and seeded at a density of
7 × 104 cells/cm2 on either 24-mm round
polycarbonate filters (pore size of 0.4 µm), 25-mm round glass
coverslips, or 30-mm culture dishes for determination of albumin
permeability, [Ca2+]i level, or MLC
phosphorylation, respectively. Experiments were performed with
confluent endothelial passage 1 monolayers 4 days after seeding.
Experimental protocols.
The basal medium used in incubations was modified Tyrode solution
(composition in mM: 150 NaCl, 2.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, and 30.0 HEPES; pH 7.4, 37°C) supplemented with
5% (vol/vol) heat-inactivated newborn calf serum (10 min,
60°C). Basal MLC phosphorylation was determined after an
initial equilibration period of 10 min. Agents were added as indicated.
Stock solution of ATP was prepared with basal medium immediately before
use. Stock solutions of calyculin A, ML-7, xestospongin C, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
acetoxymethyl ester (BAPTA)-AM were prepared with dimethyl sulfoxide
(DMSO). Appropriate volumes of these solutions were added to the cells,
yielding final solvent concentrations
0.1% (vol/vol). The same final
concentrations of DMSO were also included in all respective control
experiments. Stock solutions of all other substances were prepared in
basal medium (composition as described above). Appropriate volumes of
these solutions were added to the cells. Identical additions of basal
medium were included in all respective control experiments.
Free [Ca2+]i.
[Ca2+]i was determined with the use of the
fluorescent Ca2+ indicator fura 2. Confluent endothelial
monolayers cultured on round glass coverslips were incubated in medium
199 supplemented with 5% (vol/vol) heat-inactivated newborn calf serum
and addition of 5 µM fura 2-AM (acetoxymethyl ester of fura 2) at
20°C in the dark. After a 50-min incubation, extracellular fura 2-AM
was removed by medium change. This was followed by a 20-min incubation
period in the same medium before measurements were started. The
coverslips were then mounted in a fluorescence microscope (IX 70;
Olympus, Hamburg, Germany). [Ca2+]i was
analyzed using a TILL Photonics (Martinsried, Germany) imaging system.
During incubations, the excitation wavelength was alternated between
340 and 380 nm (bandwidth of 8 nm). Emitted light was detected at 510 nm. Fura 2 fluorescence was calibrated according to the method
described by Grynkiewicz et al. (8). For this purpose, the
cells were exposed to 5 µM ionomycin in modified Tyrode solution
containing either 3 mM Ca2+ or 5 mM EGTA to obtain the
maximum (Rmax) and the minimum (Rmin) of the
ratio of fluorescence, respectively. [Ca2+]i
was calculated according to the equation
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with use of Kd, the dissociation constant
of fura 2 (8), and
, the ratio of the 380-nm excitation
signals of ionomycin-treated cells at 5 mM EGTA and at 3 mM
Ca2+.
Determination of MLC phosphorylation.
The phosphorylation of MLC was determined by glycerol-polyacrylamide
gel electrophoresis and immunoblot analysis using an anti-MLC antibody
as described previously (17). Cells cultured on 30-mm
dishes were incubated as indicated in the text. After pretreatment, the
incubation medium was rapidly removed and the reaction was terminated
by addition of ice-cold trichloroacetic acid (1.2 mM). Precipitated
proteins were transferred into Eppendorf reaction tubes and centrifuged
for 10 min at 14,000 g at 4°C. The sediments were washed
two times with ice-cold diethyl ether. After evaporation of the ether
at room temperature, the sediments were suspended in 30 µl of lysis
buffer (8.8 M urea, 10 mM dithiothreitole, 5 mM thioglycolate, 0.6 mM
phenylmethylsulfonyl fluoride, 10 µM cantharidin, 60 mM imidazol, 20 mM Tris, and 23 mM glycine, pH 8.8). The lysate was centrifuged at
14.000 g for 5 min. Afterward, 30 µl of a saturated
sucrose-bromphenol blue solution were added and 10 µl of lysate
(20-40 µg protein) per lane were run at 400 V and 18°C on a
10% polyacrylmamide-40% glycerol gel. Before the lysates were loaded,
preelectrophoresis was performed at 400 V for 1.5 h. This
procedure allows separation of nonphosphorylated MLCs from the
phosphorylated ones, the latter of which migrate more rapidly under
this condition. Electrophoretically separated proteins were
transblotted on polyvinylidene difluoride membranes and were incubated
with an anti-MLC antibody (clone MY-21, Sigma Chemical, Deisenhofen,
Germany) followed by an alkaline phosphatase-coupled second antibody as
previously described (14). The blots were scanned, and the
percentage of MLC phosphorylation (expressed as percentage of total
MLC) was calculated from the blot areas of non- (MIC), mono- (MLC-P),
and diphosphorylated MLC (MLC ~ PP) as follows
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Because MLC can become diphosphorylated, MLC phosphorylation
varies between 0 and 200%.
Materials.
Falcon plastic tissue culture dishes were from Becton Dickinson
(Heidelberg, Germany); ATP was from Boehringer (Mannheim, Germany);
BAPTA-AM, calyculin A, ML-7, and xestospongin C were from Calbiochem
(Bad Soden, Germany); Transwell polycarbonate filter inserts (24 mm
diameter, 0.4 µm pore size) were from Costar (Bodenheim, Germany);
newborn calf serum, medium 199, penicillin-streptomycin, and
trypsin-EDTA were from GIBCO Life Technologies (Eggenstein, Germany);
fura 2-AM was from Molecular Probes (Leiden, The Netherlands); polyvinylidene difluoride was from Millipore (Eschborn, Germany); dithiothreitol, phenylmethylsulfonyl fluoride, and thioglycolate were
from Sigma Chemical. All other chemicals were of the best available
quality, usually analytical grade.
Statistical analysis.
Data are given as means ± SD of n = 6 experiments
using independent cell preparations. Statistical analysis of data was
performed according to Student's unpaired t-test.
Probability (P) values of <0.05 were considered significant.
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RESULTS |
Effect of ATP on MLC phosphorylation and
[Ca2+]i.
Addition of 10 µM ATP reduced MLC phosphorylation of aortic
endothelial monolayers from a basal level of 36 ± 4% of total MLC to 6 ± 3% within 20 min (Fig.
1). The onset of MLC dephosphorylation coincided with the ATP-induced rise in
[Ca2+]i. ATP (5-100 µM) reduced the
level of net MLC phosphorylation in a concentration-dependent manner
(Fig. 2).

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Fig. 1.
Effect of ATP on cytosolic Ca2+ concentration
([Ca2+]i) and myosin light chain (MLC)
phosphorylation of aortic endothelial monolayers. A:
increase in [Ca2+]i in response to ATP (10 µM). A representative single recording is shown. B: MLC
dephosphorylation in response to ATP (10 µM). Means ± SD of
n = 5 separate experiments of independent cell
preparations are given. At time 1 min, MLC phosphorylation was
significantly different from control (P < 0.05).
C: Western blot analysis of MLC of endothelial cells exposed
to ATP (10 µM) at time 0 and at 1, 10, 20, and 30 min. To
enhance net dephosphorylation of MLC, endothelial cells were exposed to
ML-7 (100 µM for 30 min), an inhibitor of MLC kinase. To induce net
phosphorylation of MLC, cells were exposed to calyculin A (Caly; 10 nM
for 60 min), an inhibitor of phosphatases. A blot of a representative
experiment is given. The bands represent, from top to
bottom, the nonphosphorylated (MLC), monophosphorylated
(MLC~P), and diphosphorylated (MLC~PP) MLC, respectively.
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Fig. 2.
Dose-dependent effect of ATP on endothelial MLC
phosphorylation. Data are means ± SD of n = 5 separate experiments of independent cell preparations. At time 1 min,
MLC phosphorylation was significantly different from control
(P < 0.05).
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To test whether the effect of ATP on MLC dephosphorylation is, in part,
transmitted via adenosine receptors, 8-phenyltheophylline (8-PT), an
adenosine receptor antagonist, was applied. Presence of 8-PT (10 µM)
left the reduction of MLC phosphorylation by ATP (10 µM) unchanged:
4 ± 3% after 20 min (not sigificant vs. ATP alone). 8-PT alone
had no effect on MLC phosphorylation (not shown). Addition of adenosine
(10 µM) also reduced MLC phosphorylation within 20 min to 5 ± 3%. This effect was inhibited in the presence of 8-PT (10 µM):
32 ± 5% MLC phosphorylation after 20 min in the presence of 8-PT
plus ATP. Adenosine did not change
[Ca2+]i (not shown).
Effects of Ca2+ chelation, ML-7, and
calyculin A on MLC phosphorylation in the absence of ATP.
First, it was tested whether intracellular Ca2+ chelation
influences MLC phosphorylation under basal conditions. For that
purpose, the cells were loaded with the Ca2+ chelator
BAPTA-AM (10 µM) (Fig. 3). When
endothelial cells were incubated in the presence of the chelator, MLC
became progressively dephosphorylated. This finding indicates that
under basal conditions MLC phosphorylation is regulated in a
Ca2+-dependent manner.

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Fig. 3.
Effect of intracellular Ca2+ chelation on
endothelial MLC phosphorylation under basal conditions. Endothelial
cells were incubated in an extracellular medium containing 1.2 mM
Ca2+ (control) or in the presence of 1.2 mM extracellular
Ca2+ plus 10 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA). Data are means ± SD of n = 5 separate
experiments of independent cell preparations. At time 5 min, MLC
phosphorylation was significantly different from control
(P < 0.05).
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Second, it was tested whether ML-7, an inhibitor of MLC kinase, affects
the basal state of MLC phosphorylation. Addition of ML-7 (10-100
µM) caused a dose-dependent reduction of MLC phosphorylation (Fig.
4). At 100 µM ML-7, MLC became
completely dephosphorylated within 10 min. This finding
indicates that MLC kinase contributes actively to the basal state of
MLC phosphorylation.

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Fig. 4.
Dose-dependent effect of MLC kinase inhibitor ML-7 on
endothelial MLC phosphorylation under basal conditions. A:
data are means ± SD of n = 5 separate experiments
of independent cell preparations. At time 1 min, MLC phosphorylation
was significantly different from control (P < 0.05).
B: Western blot analysis of MLC of endothelial cells exposed
to ML-7 (100 µM) at time 0 and at 1, 10, 20, and 30 min. A
blot of a representative experiment is given. The bands represent, from
top to bottom, the nonphosphorylated (MLC),
monophosphorylated (MLC~P), and the diphosphorylated (MLC~PP) MLC,
respectively.
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Third, it was tested whether an inhibitor of protein phosphatases
influences MLC phosphorylation under basal conditions. The effects of
calyculin A (1-10 nM), an inhibitor of PP-1 and PP-2A isoenzymes
(9), was analyzed. As shown in Fig.
5, the inhibitor caused an increase in
MLC phosphorylation in a dose-dependent manner. This finding indicates
that the basal state of MLC phosphorylation is also influenced by
protein phosphatase activity.

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Fig. 5.
Dose-dependent effect of protein phosphatase inhibitor
calyculin A on endothelial MLC phosphorylation under basal
conditions. Data are means ± SD of n = 5 separate experiments of independent cell preparations.
* P < 0.05 vs. control. Because MLC can become
diphosphorylated, MLC phosphorylation varies between 0 and 200% (see
Determination of MLC phosphorylation in MATERIALS AND
METHODS).
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Effect of ATP on MLC phosphorylation in the presence of
xestospongin C.
It was studied whether the ATP-induced net dephosphorylation of MLC is
triggered by the concomitant increase in the ATP-induced rise in
[Ca2+]i. To test this, endothelial cells were
incubated for 20 min in the presence of xestospongin C (10 µM), an
inhibitor of the inositol trisphosphate (IP3)-stimulated
Ca2+ release from endoplasmic reticulum, before ATP (10 µM) was added to the cells. If the ATP-induced MLC dephosphorylation
is Ca2+ independent, ATP will induce dephosphorylation of
MLC also under those conditions, which prevent the ATP-induced
Ca2+-release. As shown in Fig.
6, pretreatment with xestospongin C abolished the ATP-induced rise in [Ca2+]i but
did not affect the ATP-induced reduction of MLC phosphorylation (Fig.
7). These results indicate that the
ATP-induced MLC dephosphorylation is Ca2+ independent.

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Fig. 6.
Effect of xestospongin C (Xe C) on ATP-induced increase
of [Ca2+]i in aortic endothelial cells.
Control cells ( ) were exposed to neither ATP nor
xestospongin C. Other cells were exposed to xestospongin C (10 µM;
) for 20 min before additional administration of ATP
(10 µM; ). Results were compared with effects of ATP
alone (10 µM; ). Means ± SD of
n = 5 separate experiments of independent cell
preparations are given. * P < 0.05 vs. control.
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Fig. 7.
Effect of xestospongin C on ATP-induced endothelial MLC
phosphorylation. Control cells ( ) were exposed to
neither ATP nor xestospongin C. Other cells were exposed to
xestospongin C (10 µM; ) for 20 min before additional
administration of ATP (10 µM; ). Results were
compared with effects of ATP alone (10 µM; ).
Means ± SD of n = 5 separate experiments of
independent cell preparations are given. At time 1 min, MLC
phosphorylation in the presence of ATP alone or ATP plus
xestospongin C was significantly different from control
(P < 0.05). MLC phosphorylation in the
presence of ATP plus xestospongin C was not significantly different
compared with MLC phosphorylation in the presence of ATP alone.
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Effect of ATP on MLC phosphorylation in calyculin A-pretreated
cells.
As shown in the preceding section, ATP given alone caused a net
dephosphorylation of MLC. Now, it was analyzed whether ATP influences
MLC phosphorylation also in the presence of a protein phosphatase
inhibitor, i.e., 10 nM calyculin A. ATP (10 µM) was added 15 min
after the addition of calyculin A, when MLC phosphorylation had started
to rise, indicating the inhibitory action of calyculin A (Fig.
8). In contrast to its effect when given
alone, ATP enhanced MLC phosphorylation in cells pretreated with a
phosphatase inhibitor. The results indicate that ATP also activates a
MLC kinase activity that only becomes apparent, however, when the
predominant phosphatase activation is prevented.

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Fig. 8.
Influence of ATP on endothelial MLC phosphorylation in
the presence of protein phosphatase inhibitor calyculin A. Control
cells ( ) were exposed to neither ATP nor calyculin A. Endothelial cells were exposed to calyculin A (10 nM;
). Where indicated, ATP (10 µM; ) was
added to the calyculin A-treated cells. Data are means ± SD of
n = 5 separate experiments of independent cell
preparations. At time 10 min, MLC phosphorylation in the presence of
calyculin A alone was significantly different from control
(P < 0.05). * P < 0.05 vs.
calyculin A alone. Because MLC can become diphosphorylated, MLC
phosphorylation varies between 0 and 200% (see Determination of
MLC phosphorylation in MATERIALS AND METHODS).
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Effect of ATP plus calyculin A on MLC phosphorylation in
Ca2+-depleted cells.
It was tested whether the ATP-induced stimulation of MLC
phosphorylation in the presence of calyculin A depends on
[Ca2+]i. For this purpose, endothelial cells
were incubated in the presence of both BAPTA and calyculin A (Fig.
9). Under these conditions, ATP no longer
increased MLC phosphorylation, indicating that the MLC kinase activity
stimulated by ATP is Ca2+ dependent.

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Fig. 9.
Influence of ATP on endothelial MLC phosphorylation in
the presence of BAPTA and calyculin A. , Control cells.
In a set of experiments, endothelial cells were loaded with BAPTA (10 µM; ) for 20 min as in Fig. 3, and then ATP (10 µM;
), calyculin A (10 nM; ), or ATP and
calyculin A ( ) were added. Data are means ± SD of
n = 5 separate experiments of independent cell
preparations. At time 10 min, MLC phosphorylation in the presence of
BAPTA alone was significantly different from control (P < 0.05). At time 20 min, MLC phosphorylation in the presence of
BAPTA + ATP, BAPTA + calyculin A or BAPTA + ATP + calyculin A was not significantly different compared with MLC
phosphorylation in the presence of BAPTA alone (P > 0.05).
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DISCUSSION |
This is the first study to analyze the effect of the physiological
mediator ATP on the phosphorylation state of MLC, the regulatory protein of the contractile machinery in endothelial cells. The major
finding is that ATP simultaneously stimulates a functional antagonism
toward phosphorylation and dephosphorylation of MLC, in which the
dephosphorylation of MLC prevails. Protein kinase activation is caused
by the rise in cytosolic Ca2+ elicited by ATP; activation
of MLC dephosphorylation is a Ca2+-independent effect of ATP.
Under basal culture conditions, MLC of porcine aortic endothelial cells
was found to be partially phosphorylated. Exposure of these cells to an
inhibitor of MLC kinase (ML-7) quickly reduced the extent of MLC
phosphorylation. This indicates that under basal conditions, MLC kinase
is one of the factors determining the state of MLC phosphorylation. The
observation that intracellular Ca2+ chelation can mimic the
effect of ML-7 indicates that the Ca2+/calmodulin-dependent
MLC kinase is involved in phosphorylation of MLC under basal
conditions. Exposure of the endothelial cells to the specific inhibitor
of the protein phosphatases PP-1 and PP-2A, calyculin A, resulted in a
rapid increase in the phosphorylation state of MLC. This result shows
that under basal conditions the level of MLC phosphorylation is also
controlled by protein phosphatases. Under basal conditions, MLC
phosphorylation is thus in a steady state in which the action of MLC
kinase is balanced by myosin phosphatases.
Exposure of endothelial cells to ATP caused a fast reduction of MLC
phosphorylation. The ATP metabolite adenosine also induced dephosphorylaion of MLC. However, the effect of ATP is not mediated by
its metabolite, as the action of adenosine but not the one of ATP can
be blunted by the adenosine receptor inhibitor 8-PT.
ATP acts on MLC phosphorylation in a dose-dependent manner. This effect
persisted for over 20 min. When protein phosphatases were first
inhibited by calyculin A and ATP was then applied, it caused an
(additional) increment in MLC phosphorylation instead of
dephosphorylation. This latter experiment demonstrates that ATP also
stimulates a protein kinase acting on the MLC. The finding that ATP,
when applied alone, causes a net dephosphorylation must therefore be
explained by a strong activation of protein phosphatases overruling the
activation of protein kinase. It was tested whether the activation of
protein phosphatase, acting on MLC, was Ca2+ dependent. The
inhibitor of IP3-regulated Ca2+ release,
xestospongin C (5, 15), was used to abolish the ATP-induced rise in [Ca2+]i. Under these
conditions, a dephosphorylation of MLC in response to ATP was still
observed. This shows that the calyculin A-sensitive protein
phosphatases activated by ATP are Ca2+ independent. This is
in accordance with previous findings that these endothelial cells
express constitutively the Ca2+-independent phosphatase
isoforms PP-1 and PP-2A (3, 10, 24), which are
specifically inhibited by calyculin A at 10 nM (9).
Recently, Verin et al. (22) found that endothelial cells can also express the (inducible) Ca2+/calmodulin-dependent
protein phosphatase type 2B, which can contribute to the control of MLC
dephosphorylation in the presence of thrombin.
It was also tested whether the protein kinase activated by ATP depends
on the ATP-induced Ca2+ rise. For this purpose, endothelial
cells were first Ca2+ depleted and then ATP and calyculin A
were added simultaneously. The fact that, under this condition, ATP no
longer caused an increase in MLC phosphorylation shows that the
ATP-induced activation of protein kinase is indeed dependent on the
Ca2+ rise. ATP stimulates both a phosphorylation of MLC by
a Ca2+-dependent activation of MLC kinase and a
dephosphorylation of MLC by Ca2+-independent protein
phosphatases. The dephosphorylation prevails.
In endothelial cells, as in cells from other tissues, little is known
about signal transduction leading to activation of myosin phosphatases.
Activation of PP-1 and PP-2A could be due to reduction of basal Rho
kinase activation, since the latter is part of an inhibitory mechanism
(4). In smooth muscle cells, PP-1 and PP-2A are also
activated via protein kinase A or G (1, 21), by mechanisms
that have not been fully analyzed. Further work is required to clarify
whether ATP activates myosin phosphatase through one of these or yet
another mechanism.
The results of this study are of particular interest for two main
reasons. First, they describe a novel effect of ATP on endothelial cells. ATP is an important vascular mediator. Its extracellular concentration is normally kept low by ectonucleotidases (7, 28) but may increase substantially at the sites of thrombus formation (11) in hypoxic myocardium (2) or
close to ATP-releasing nerve endings. The present findings also explain
why ATP was found to reduce endothelial barrier permeability despite a
cytosolic Ca2+ rise (16). Second, ATP is one
of the few known physiological mediators and the first described for
endothelial cells that strongly induces MLC dephosphorylation. The
results of this study indicate that this effect is due to an activation
of myosin phosphatases. The vast majority of other physiological
mediators investigated in smooth muscle cells acts through an
inhibition mechanism if affecting myosin phosphatases. The
identification of a physiological mechanism of myosin phosphatase
activation is of particular interest for endothelial pathophysiology as
its knowledge may lead to new therapeutic principles for stabilizing
the endothelial barrier by dephosphorylation of endothelial MLC.
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ACKNOWLEDGEMENTS |
This work was supported by the Deutsche Forschungsgemeinschaft,
grant A3 and A4 of SFB 547.
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FOOTNOTES |
This work is a part of the thesis submitted by U. Schavier-Schmitz.
Address for reprint requests and other correspondence: T. Noll,
Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen Germany (E-mail:
thomas.noll{at}physiologie.med.uni-giessen.de).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 17 December 1999; accepted in final form 29 March 2000.
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