Store-operated calcium entry and increased endothelial cell
permeability
Natalie
Norwood1,
Timothy M.
Moore1,
David A.
Dean2,
Rakesh
Bhattacharjee1,
Ming
Li1, and
Troy
Stevens1
Departments of 1 Pharmacology and 2 Microbiology,
University of South Alabama College of Medicine, Mobile, Alabama
36688
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ABSTRACT |
We hypothesized that myosin light chain kinase
(MLCK) links calcium release to activation of store-operated calcium
entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to
activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin
stabilization eliminated the thapsigargin-induced current. Thapsigargin
increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium
entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not
prevent thapsigargin from increasing permeability. Rather, inhibition
of MLCK activity increased permeability that was especially prominent
in low extracellular calcium. In conclusion, MLCK links store depletion
to activation of a store-operated calcium entry channel. However,
inhibition of calcium entry by MLCK is not sufficient to prevent
thapsigargin from increasing endothelial cell permeability.
lung; myosin light chain kinase; signal transduction; inositol
trisphosphate; capacitative calcium entry
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INTRODUCTION |
MAJNO AND PALADE
(16) originally suggested that inflammatory mediators
stimulate endothelial cell retraction necessary to increase
permeability. General support for this hypothesis continues today as
the molecular events that control barrier function are examined
(15, 17, 18). Recent measurements indicate that endothelial cells possess a constitutive inward tension resulting from
the interaction of F-actin with nonmuscle myosin that forms an
actomyosin complex (10, 26, 27). Actomyosin interaction is
stimulated by reversible phosphorylation of 20-kDa myosin light chain
(MLC20). An endothelial cell-specific MLC kinase (MLCK) is
the primary isoform that regulates phosphorylation of MLC20 (34, 35). Gq-coupled agonists like histamine
and thrombin activate MLCK, which increases MLC20
phosphorylation from its constitutive level of
0.4 to
1.2 mol
phosphate/mol MCL20 and further promotes centripetally
directed tension (10, 19, 20, 27, 38, 39).
Although a central role for MLCK in endothelial cell barrier function
has been demonstrated, the precise relationship between MLCK-induced
retraction and generation of intercellular gaps is not fully
established. MLCK activation is clearly linked to increased permeability, and inhibition of MLCK reduces permeability evoked by
Gq-coupled agonists (9, 19, 27).
However, direct inhibition of cell-cell and cell-matrix tethering under
conditions of constitutive MLC20 phosphorylation is
sufficient to increase permeability. Furthermore, MLCK may play a
secondary or alternate role in regulating the endothelial barrier
response by inhibiting cytosolic calcium concentration
([Ca2+]i) responses to neurohumoral
inflammatory agonists (11, 36, 37). Thus the specific
function of MLCK in linking cell activation to increased permeability
is not completely understood.
An elevation in [Ca2+]i associated with
activation of store-operated calcium entry is sufficient to increase
endothelial cell permeability (4, 13, 18, 28, 29).
Activation of store-operated calcium entry occurs after depletion of
intracellular calcium stores either by stimulation of calcium release
(e.g., histamine or thrombin) or by inhibition of calcium reuptake
(e.g., thapsigargin) into storage sites. Although the mechanism linking
store depletion to activation of calcium entry is unknown, a
conformational or physical coupling model has previously been proposed
(1, 24). The original hypothesis suggested that a decrease
in stored calcium alters the inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3] receptor conformation that directly opens a membrane calcium channel. The possibility that cytoskeletal elements tether intracellular
organelles or the Ins(1,4,5)P3
receptor to membrane channel function has also been considered
(3, 12, 23). An extension of this latter possibility is
that intracellular organelles possessing calcium stores (e.g.,
endoplasmic reticulum) or the
Ins(1,4,5)P3 receptor are coupled
to store-operated calcium entry channels through the cytoskeleton that
is held under tension. Thus changes in MLCK-dependent tension may
directly regulate activation of store-operated calcium entry,
suggesting that MLCK may influence endothelial cell barrier function by
controlling calcium responses to Gq agonists. Our present
studies tested the hypothesis that MLCK activation by inflammatory
calcium agonists regulates calcium entry that is important for control
of endothelial cell barrier function.
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METHODS |
Measurement of [Ca2+]i.
Rat pulmonary artery endothelial cells (RPAECs) were isolated and
cultured for study at passages 9-17. For
calcium measurements, the cells were seeded at ~1.5 × 105 cells/ml on two-chambered glass coverslips (Nalge Nunc
International) and grown to confluence in serum-containing medium
continuously for 4-7 days without a change in medium. Cells were
loaded with a fura 2-AM loading buffer (2 ml of Krebs buffer with 25 mM
HEPES plus 2 mM or 100 nM calcium, 3 mM fura 2-AM, and 6 µl of
Pluronic acid) for 20 min in a CO2 incubator at 37°C. The
cells were then washed with 2 ml of Krebs buffer and treated with
deesterification medium (2 ml of Krebs buffer with 25 mM HEPES plus 2 mM or 100 nM calcium) for an additional 20 min. After deesterification, [Ca2+]i was assessed with an Olympus IX70
inverted microscope at ×400 with a xenon arc lamp photomultiplier
system (Photon Technologies, Monmouth Junction, NJ), and data were
acquired and analyzed with PTI Felix software. Epifluorescence was
measured from three to four endothelial cells in a confluent monolayer,
and the changes in [Ca2+]i are expressed as
the fluorescence ratio of Ca2+-bound (340-nm) to
Ca2+-unbound (380-nm) excitation wavelengths (ratio
340/380) emitted at 510 nm. In vitro calibrations were then performed
with the fura 2 calcium imaging calibration kit (Molecular Probes).
Microinjections.
RPAECs were trypsin dispersed on etched glass coverslips placed in
60-mm plastic culture dishes. The cells were allowed to reattach for at
least 24 h in serum-containing growth medium. Approximately
75-100 cells in small confluent patches were then microinjected
with glass capillary pipettes pulled with a pipette puller (World
Precision Instruments, Sarasota, FL) and a Narishige micromanipulator.
Heparin (5 U/ml) in phosphate-buffered saline was microinjected.
Approximately 3 × 10
10 ml was injected into each
cell (5).
Electrophysiology.
Whole cell patch clamp was utilized to measure transmembrane ion flux
in thapsigargin-stimulated RPAECs according to previously described
methods (18). Confluent RPAECs were enzyme dispersed, seeded onto 35-mm plastic culture dishes, and then allowed to reattach
for at least 24 h before the patch-clamp experiments. Single
RPAECs exhibiting flat polyhedral morphology were studied. These cells
were chosen for study because their morphology was consistent with
RPAECs from a confluent monolayer. These single cells have previously
been shown to possess electrophysiological recordings generally similar
to those observed in confluent monolayers (28). The
extracellular solution was composed of (in mM) 110 tetraethylammonium
aspartate, 10 calcium aspartate, 10 HEPES, and 0.5 3,4-diaminopyridine;
and the pipette solution was composed of (in mM) 130 N-methyl-D-glucamine, 1.15 EGTA, 10 HEPES, and 1 Ca(OH)2 with and without 2 Mg2+-ATP. Both
solutions were adjusted to 290-300 mosM with sucrose and pH 7.4 with methane sulfonic acid. [Ca2+]i was
estimated as 100 nM (5a). The pipette resistance was 2-5 M
. Data were obtained with a HEKA EPC9 amplifier (Lambrecht/Pfaltz) and sampled on-line with Pulse+Pulsefit software (HEKA) All recordings were made at room temperature (
25°C). To generate current-voltage relationships, voltage pulses were applied from
100 to +100 mV in
20-mV increments, with 200-ms duration for each voltage step and a 2-s
interval between steps. The holding potential between each step was 0 mV. The experimental protocols were established as follows:
1) vehicle control (DMSO in patch pipette; n = 10 experiments), 2) thapsigargin control (1 µM
thapsigargin in patch pipette; n = 15 experiments),
3) ML-9 plus thapsigargin (15 µM ML-9 pretreatment for
10-30 min, thapsigargin in patch pipette; n = 13 experiments), 4) jasplakinolide plus thapsigargin (1 µM jasplakinolide pretreatment for 4 h, thapsigargin in patch
pipette; n = 4 experiments), and 5)
cytochalasin D (10 mM cytochalasin D in patch pipette;
n = 10 experiments).
Estimation of diffusive capacity.
RPAECs were seeded onto Transwell inserts (6.5-mm diameter, 0.4-mm pore
size; Costar) at a density of 8.5 × 105 cells/ml in a
final volume of 100 µl of DMEM plus 10% FBS. The inserts were placed
into 24-well plates containing 600 µl of growth medium, and the cells
were allowed to grow for 5 days with one change of medium. After
confluence was achieved, the growth medium in the upper chamber was
replaced with 100 µl of a 1 mg/ml FITC-dextran (mol wt 10,000)
solution in Krebs-Henseleit physiological salt solution (PSS). The
insert was then moved to a fresh lower well containing 600 µl of PSS.
The cells were equilibrated with these solutions at 37°C in a
CO2 incubator for 10 min. After equilibration, the
Transwell insert was placed into another lower chamber containing 600 µl of PSS, and the FITC-dextran was allowed to diffuse across the
monolayer for 30 min. This procedure was repeated three times so that a
total time of 2 h for assessing monolayer integrity was employed.
Samples from the lower chamber (50 µl) were taken in triplicate and
placed in 96-well cluster plates for measuring fluorescent intensity
(Perkin-Elmer luminescence spectrometer LS 50B) with an excitation of
480 nm and an emission at 530 nm. Fluorescence values were then
converted to milligrams of FITC-dextran per milliliter with a standard
curve that was generated concurrent with the measurements of monolayer
integrity. With these values, diffusive capacity (PS) was calculated by
determining the net rate of FITC-dextran flux
(Js) generated for each concentration difference
(
C) across the monolayer with the equation PS = Js/(
C). PS is expressed in nanoliters per minute.
Statistical methods.
Data are reported as means ± SE. Comparisons were made with
either unpaired Student's t-test or one-way analysis of
variance with repeated measures as appropriate. A Student-Newman-Keuls post hoc test was applied. Differences were considered significant at
P < 0.05.
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RESULTS |
Regulation of calcium release by MLCK.
Ins(1,4,5)P3 receptors possess
putative ankyrin binding domains predicted to associate the receptor
with cytoskeletal elements held under tension in endothelial cells
(3, 7, 20, 38, 39). We therefore tested whether inhibiting
MLCK, which has previously been shown to decrease endothelial cell
tension, would alter the kinetics of calcium release. Figure
1 demonstrates that acute application of
the MLCK inhibitor ML-9 to confluent fura 2-AM-loaded RPAECs produced a
transient increase in [Ca2+]i in the presence
(2 mM; Fig. 1A, Table
1) and relative absence (100 nM;
Fig. 1B) of extracellular calcium, indicating that ML-9 stimulates calcium release. Similar results were obtained with other
MLCK inhibitors including W-7, which caused a peak increase in
[Ca2+]i from baseline values of 137 ± 2 nM (ratio 340/380 = 0.9 ± 0.01) to 1.8 ± 0.86 (ratio
340/380; P < 0.05; n = 9 experiments).
The protein kinase (PK) A inhibitor H-89 did not alter
[Ca2+]i at concentrations specific for PKA
but increased [Ca2+]i when used at
concentrations that reportedly inhibit MLCK (Fig. 1C).
Similarly, inhibition of PKC activity with chelerythrine did not alter
[Ca2+]i (data not shown). These data
therefore implicate MLCK, but not PKA or PKC, in the regulation of
calcium release in RPAECs.

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Fig. 1.
Acute inhibition of myosin light chain kinase (MLCK)
increased cytosolic Ca2+ concentration
([Ca2+]i) in rat pulmonary artery endothelial
cells (RPAECs). A: 100 µM ML-9 applied directly to
confluent RPAECs incubated in an extracellular Ca2+
concentration ([Ca2+]o) of 2 mM produced a
transient increase in [Ca2+]i that returned
to baseline levels (n = 5 experiments).
P < 0.05. B: reducing
[Ca2+]o from 2 mM to 100 nM did not prevent
ML-9 from increasing [Ca2+]i
(n = 8 experiments). P < 0.05. C: representative trace showing that application of H-89 to
RPAECs at a concentration that specifically inhibits protein kinase A
activity (50 nM) did not increase [Ca2+]i,
but addition of H-89 at a concentration that also inhibits MLCK
activity (30 µM) transiently increased
[Ca2+]i. Ratio 340/380, ratio of 340- to
380-nm fluorescence.
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Because the Ins(1,4,5)P3-sensitive
calcium pool is depleted after sarcoplasmic/endoplasmic reticulum
Ca2+-ATPase inhibition (31), we utilized
thapsigargin to assess whether ML-9 increased
[Ca2+]i by stimulating calcium release from
an Ins(1,4,5)P3-sensitive calcium
pool. Although application of thapsigargin in a low extracellular Ca2+ concentration ([Ca2+]o)
produced a transient increase in [Ca2+]i from
0.7 ± 0.02 to 1.6 ± 0.13 (ratio 340/380; P < 0.05; n = 7 experiments), pretreatment of the cells
incubated in 2 mM [Ca2+]o with ML-9 nearly
abolished the thapsigargin-induced calcium release [from 0.9 ± 0.03 to 1.1 ± 0.05 (ratio 340/380); P = not significant (NS) from baseline]. Similarly, thapsigargin pretreatment prevented ML-9 from increasing [Ca2+]i (ratio
340/380 = 0.7 ± 0.03; P = NS from
baseline; n = 5 experiments), suggesting that ML-9 and
thapsigargin target a similar
Ins(1,4,5)P3-sensitive pool. To
confirm this possibility, RPAECs were microinjected with heparin to
inhibit the Ins(1,4,5)P3 receptor
(30). In physiological concentrations of
[Ca2+]o, baseline
[Ca2+]i ratios were similar in
heparin-injected (ratio 340/380 = 0.9 ± 0.03;
n = 6 cells) and PBS-injected or noninjected cells
(ratio 340/380 = 0.9 ± 0.03; n = 5 cells),
demonstrating good cell viability. In control cells, application of
ML-9 increased [Ca2+]i to 1.6 ± 0.6 (ratio 340/380; P < 0.05; n = 6 cells)
and the subsequent addition of thrombin resulted in an abrupt peak
increase in [Ca2+]i to 3.4 ± 0.5 (ratio
340/380; P < 0.05; n = 6 cells; Fig.
2A). Heparin microinjection
prevented both the ML-9- and thrombin-induced increase in
[Ca2+]i (Fig. 2, B and
C). Thus these data demonstrate that ML-9 promotes calcium
release through the Ins(1,4,5)P3
receptor.

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Fig. 2.
Inhibition of MLCK causes calcium release from inositol
1,4,5-trisphosphate
[Ins(1,4,5)P3]-sensitive calcium
pools. A: representative trace demonstrating that
application of 15 µM ML-9 increased
[Ca2+]i. Subsequent addition of thrombin (TH;
7 U/ml) produced a characteristic spike in
[Ca2+]i due to calcium release from
Ins(1,4,5)P3-sensitive calcium
pools. B: treatment of heparin (5 U/ml)-microinjected cells
with ML-9 (100 µM) and thrombin (7 U/ml) exhibited significantly
diminished [Ca2+]i responses. C:
average peak [Ca2+]i responses to ML-9 and
thrombin, demonstrating that heparin microinjection significantly
diminished Ins(1,4,5)P3-dependent
sensitivity (n = 6 experiments). P < 0.05.
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Role of MLCK in activation of store-operated calcium entry.
MLCK regulates actomyosin interaction and the inward centripetal
tension in endothelial cells, although it is unclear whether MLCK-dependent cell tension effects activation of
store-operated calcium entry. We therefore next examined whether
inhibition of MLCK regulates store-operated calcium entry
(37). Figure 3A demonstrates that thapsigargin produced a slowly developing, sustained increase in [Ca2+]i. However, ML-9
pretreatment significantly attenuated thapsigargin-dependent calcium
release (ratio 340/380 = 1.6 ± 0.1 without ML-9,
n = 7 experiments, vs. ratio 340/380 = 0.2 ± 0.05 with ML-9, n = 8 experiments; P < 0.05) and abolished the calcium entry response.
Because ML-9 reduced thapsigargin-dependent calcium release, the
calcium stores were likely not depleted, suggesting inhibition of
calcium entry could be due to either preservation of the intracellular
calcium pool or disruption of a mechanism gating the store-operated
calcium entry channel. To address this issue, thapsigargin was applied first to activate store-operated calcium entry and then ML-9 was added
(Fig. 3C). Addition of ML-9 immediately reduced
[Ca2+]i, suggesting that MLCK regulates the
activation state of a membrane channel. To confirm this idea,
thapsigargin was applied to RPAECs incubated in nominally calcium-free
medium (100 nM) followed by readdition of extracellular calcium (2 mM).
Thapsigargin stimulated a transient, calcium release-dependent increase
in [Ca2+]i. Replenishing
[Ca2+]o resulted in a sustained, calcium
entry-dependent increase in [Ca2+]i that was
immediately reduced after application of ML-9 (Fig. 3D).

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Fig. 3.
Inhibition of MLCK inactivates store-operated calcium entry.
A: thapsigargin (TG; 1 µM) produced a slowly developing,
sustained increase in [Ca2+]i
(n = 6 experiments). B: ML-9 (100 µM)
pretreatment caused a transient increase in
[Ca2+]i and prevented thapsigargin (1 µM)-induced calcium release and activation of store-operated calcium
entry (n = 5 experiments). C: activation of
store-operated calcium entry with 1 µM thapsigargin was immediately
reduced after application of ML-9 (100 µM; n = 4 experiments). D: application of thapsigargin (1 µM) to
RPAECs incubated in 100 nM [Ca2+]o produced a
transient increase in [Ca2+]i that returned
to baseline value. Restoring [Ca2+]o to 2 mM
resulted in a rapid and sustained increase in
[Ca2+]i because calcium entry occurs through
store-operated calcium entry channels. ML-9 (15 µM) immediately
inactivated store-operated calcium entry and returned
[Ca2+]i to near baseline levels
(n = 6 experiments).
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The ML-9-dependent decrease in [Ca2+]i
observed in Fig. 3, C and D, could be due to
inhibition of a calcium entry channel, stimulation of calcium
extrusion, or membrane depolarization. We addressed this issue using
whole cell electrophysiology in single RPAECs in voltage-clamp mode.
Solutions were designed to isolate a store-operated calcium entry
current as Moore et al. (18) have previously
described. Although cells exhibited only a slight leak current under
unstimulated conditions, inclusion of thapsigargin in the patch pipette
activated a calcium current that at positive voltages was inwardly
rectifying (Fig. 4, A and
B). This current was similar to the previous report by Moore
et al. in these cells. Furthermore, exclusion of Mg-ATP from the patch
pipette abolished the store-operated calcium entry current (Fig.
4C). Similarly, pretreatment with ML-9 to cells supplied
with ATP abolished the response to thapsigargin (Fig. 4D),
suggesting that MLCK regulates a calcium entry channel likely due to a
phosphorylation-dependent event.

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Fig. 4.
Thapsigargin activation of a store-operated calcium entry current
is dependent on MLCK. Shown are representative traces of current
voltage (I-V) plots at a voltage range of 100
to +100 mV. A: voltage-clamped cells exhibited only a slight
leak current under unstimulated conditions. B: inclusion of
1 µM thapsigargin in the patch pipette induced an inward calcium
current that at positive voltages was inwardly rectifying.
C: excluding Mg-ATP from the internal solution prevented
thapsigargin (1 µM) from activating the store-operated calcium entry
current. D: pretreating RPAECs with 15 µM ML-9 for 10 min
similarly prevented thapsigargin (1 µM) from activating an inward
calcium current.
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Activation of MLCK evokes enzyme translocation from the cytosol to the
F-actin cytoskeleton where it promotes actomyosin interaction and
increases centripetally directed tension (9, 10, 20, 34,
38). We therefore examined whether the configuration of F-actin
represented an important determinant of store-operated calcium entry.
Initial studies utilized cytochalasin D to disrupt F-actin filaments
and eliminate RPAEC inward tension (Fig.
5). Cytochalasin D activated an inward
calcium current with biophysical properties resembling a
thapsigargin-sensitive store-operated calcium entry current (Table
2). To further address this idea, jasplakinolide was utilized to stabilize F-actin. Pretreatment with
jasplakinolide eliminated the response to thapsigargin, suggesting that
MLCK may gate store-operated calcium entry channels through the
regulation of actomyosin-based tension.

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Fig. 5.
F-actin regulation of store-operated calcium entry. Shown
are representative traces from I-V plots at a
voltage range of 100 to +100 mV. Inclusion of cytochalasin D (Cyt D;
10 µM) in the patch pipette activated an inward calcium current that
was inwardly rectifying at positive voltages. Stabilization of F-actin
with jasplakinolide (1 µM) prevented thapsigargin (1 µM) from
activating a store-operated calcium entry current.
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We performed studies to address whether the action of ML-9 was
due to its inhibitory effect on MLCK. Figure
6, A and B,
demonstrates the dose-dependent inhibition of store-operated calcium
entry by ML-9, with an IC50 of 4.6 µM, similar to its
calculated IC50 for MLCK (3.8 µM). Moreover, peak
[Ca2+]i responses to thapsigargin are plotted
in Fig. 6C and demonstrate that although inhibition
of PKA or PKC does not alter the [Ca2+]i
response to thapsigargin, inhibition of MLCK with ML-9, W-7, or H-89
each significantly reduced the [Ca2+]i
response to thapsigargin. Thus it is most likely that the effect of
ML-9 is through its inhibition of MLCK activity.

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Fig. 6.
Store-operated calcium entry is regulated by MLCK.
A: pretreatment with ML-9 produced a dose-dependent
inhibition of the calcium response to thapsigargin. B:
utilizing the [Ca2+]i response to
thapsigargin as in A, the calculated IC50 of
ML-9 is 4.6 µM, similar to its direct IC50 for MLCK (3.8 µM). C: peak response to thapsigargin was attenuated with
inhibitors of MLCK, including ML-9 (100 µM), W-7 (calcium/calmodulin
antagonist; 500 µM), and H-89 (30 µM; n = 5 experiments/group). P < 0.05 vs. thapsigargin.
However, inhibition of neither protein kinase A with H-89 (50 nM) nor
protein kinase C with chelerythrine (CHL; 0.66 µM) prevented
thapsigargin from increasing [Ca2+]i
(n = 4 experiments/group). P = not
significant (NS) compared with thapsigargin. * Significantly
different from control, P < 0.05.
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MLCK and endothelial cell permeability.
Inhibition of MLCK activity prevents the increase in RPAEC permeability
induced by Gq-coupled agonists like thrombin, although it
is unclear whether MLCK inhibition has similar salutary effects on
permeability changes induced by activation of store-operated calcium
entry. RPAEC monolayers exhibited a constitutive diffusive capacity to
a 23-Å FITC-dextran (mol wt 10,000) tracer that increased 27% after
application of thapsigargin (Fig.
7A). Although the increase in
permeability was greatest 30 min after the application of thapsigargin,
only a slight increase in permeability was apparent 2 h after
treatment, suggesting that barrier function improved over the time
course evaluated (Fig. 7B). Consistent with our previous
reports (4, 13, 18, 29), the thapsigargin-induced increase in
permeability required 2 mM [Ca2+]o,
indicating that activation of store-operated calcium entry was the
stimulus for barrier disruption (Fig. 7, C and
D).

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Fig. 7.
Calcium entry is required for thapsigargin to increase RPAEC
permeability. A: confluent RPAEC monolayers exhibited
constitutive flux to a FITC-dextran (mol wt 10,000) tracer that was
increased with thapsigargin (1 µM; n = 24 experiments). Measurements were made at 1 h. PS, diffusive
capacity. P < 0.05. B: when evaluated over
a 2-h time course, control monolayers exhibited similar, stable rates
of macromolecular flux (Js). The
thapsigargin-induced increase in permeability was greatest at the
30-min time point and recovered toward control value by 2 h
(n = 24 experiments), indicating a reduction in barrier
diffusive capacity. P = NS vs. control.
C: in 100 nM [Ca2+]o, thapsigargin
(1 µM) did not increase permeability (n = 4 experiments). Measurements were made at 1 h. P = NS vs. control. D: in 100 nM
[Ca2+]o, both control and
thapsigargin-treated cells exhibited similar diffusive capacities over
a 2-h time course (n = 4 experiments).
P = NS. * Significantly different from
control, P < 0.05.
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Because ML-9 inhibited calcium entry, we tested its effect on
thapsigargin-induced barrier disruption. In the presence of 2 mM
[Ca2+]o, ML-9 produced a 20% increase in
endothelial cell permeability and did not prevent thapsigargin from
further increasing diffusive capacity by 45% (Fig.
8A). Both ML-9 and
thapsigargin plus ML-9 treatments increased permeability to the
greatest extent at 30 min (Fig. 8B). Permeability induced by
ML-9 recovered by 60 min. However, permeability induced by treatment of
thapsigargin plus ML-9 did not fully recover, suggesting that MLCK
activity is required for restoration of barrier function. Sensitivity
to ML-9 was increased when studies were conducted with low
[Ca2+]o. In the presence of 100 nM
[Ca2+]o, ML-9 increased RPAEC permeability by
108% (Fig. 8C); this increase in permeability did not
reverse (Fig. 8D). Similar to our prior studies (4, 13, 18,
29), however, a further thapsigargin-induced increase in
permeability was prevented by reducing the electrochemical membrane
calcium gradient (Fig. 8, C and D). Thus the data
support the idea that in low [Ca2+]o MLCK is
barrier protective.

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Fig. 8.
Inhibition of MLCK increases permeability when
[Ca2+]o is reduced to 100 nM. A:
in 2 mM [Ca2+]o, ML-9 (100 µM) slightly
increased RPAEC diffusive capacity to the FITC-dextran (mol wt 10,000)
tracer (n = 15 experiments). P < 0.05. However, ML-9 did not prevent thapsigargin (1 µM) from further
increasing permeability (n = 10 experiments).
P = NS. Measurements were made at 1 h. B:
both ML-9 (100 µM) and the combined treatment of ML-9 (100 µM) with
thapsigargin (1 µM) produced their greatest increase in permeability
at 30 min (n = 15-24 experiments/group).
P < 0.05. The increase in permeability was reduced by
2 h, indicating a reduction in diffusive capacity. ML-9-induced
permeability increased returned to control values by 90 min.
P = NS vs. control. C: in 100 nM
[Ca2+]o, ML-9 (100 µM) produced a large
increase in permeability. Thapsigargin did not further increase
permeability (n = 4-8 experiments/group).
Measurements were made at 1 h. P = NS vs.
ML-9. D: evaluation of permeability over a 2-h time
course revealed minimal restoration of diffusive capacity, suggesting
that ML-9-induced permeability did not reverse (n = 8 experiments/group). P < 0.05. * Significantly
different from control, P < 0.05.
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DISCUSSION |
Although activation of MLCK promotes endothelial cell
permeability, the link between enzyme activation and generation of
intercellular gaps is incompletely understood. Similarly, activation of
store-operated calcium entry is sufficient to increase endothelial cell
permeability, but the mechanism(s) responsible for activation of the
membrane channel is unknown. Our present studies tested the hypothesis that MLCK activation by inflammatory calcium agonists regulates calcium
entry important for control of endothelial cell barrier function.
The MLCK inhibitor ML-9 was utilized to assess kinase regulation of
endothelial cell store-operated calcium entry and barrier function.
ML-9 inhibits ATP binding to MLCK, with an IC50 of 3.8 µM, similar to its IC50 for regulation of store-operated
calcium entry. Moreover, neither PKA nor PKC inhibitors influenced
store-operated calcium entry, suggesting that ML-9 did not alter
[Ca2+]i responses through either of these
kinases. Our findings, however, cannot rule out the possibility that
ML-9 inhibits other, currently unidentified kinases in addition to MLCK.
Calcium signaling.
Initial studies utilizing ML-9 indicated that it induces a transient
rise in [Ca2+]i due to calcium
release from a thapsigargin- and heparin-sensitive intracellular store.
Two known intracellular actions of heparin could account for these
observations (30). The most likely effect and most widely
accepted action of heparin are via its direct binding to the
Ins(1,4,5)P3 receptor at the
Ins(1,4,5)P3 binding site. In this
context, our data suggest that ML-9 alters gating characteristics of
the Ins(1,4,5)P3 receptor in its
constitutive environment, under basal levels of
[Ca2+]i,
Ins(1,4,5)P3, and calmodulin
(2, 21), to transiently increase calcium permeability.
However, heparin may also uncouple receptor activation of
Gq proteins and thus interrupt
Ins(1,4,5)P3 production. In this
context, our data suggest that ML-9 could stimulate
Ins(1,4,5)P3 production. We do not
currently know whether ML-9 alters inositol polyphosphate metabolism.
Future studies will be required to more completely address how heparin
specifically inhibits calcium release from the
Ins(1,4,5)P3 receptor.
Recent studies (11, 36, 37) have indicated that ML-9,
likely by inhibiting MLCK activity, prevents activation of
store-operated calcium entry. Our data support these previous findings,
although we observed that ML-9 reduced both thapsigargin-stimulated
calcium release and calcium entry. This finding suggested that ML-9 may prevent activation of store-operated calcium entry by interfering with
the ability of thapsigargin to deplete the calcium store. We therefore
conducted studies in which thapsigargin was utilized to first activate
store-operated calcium entry before ML-9 was applied. Under these
conditions, ML-9 immediately reduced [Ca2+]i,
consistent with the idea that MLCK influences the activation state of a
membrane calcium channel, although stimulation of calcium extrusion
through the plasmalemmal Ca2+-ATPase or
Na+/Ca2+ exchanger could not be eliminated.
To further address whether MLCK activity regulates a membrane calcium
channel directly, patch-clamp studies were undertaken in which
stimulation of calcium entry currents could be specifically studied
without the confounding influence of calcium extrusion mechanisms.
Although thapsigargin stimulated a calcium entry current similar to
that in these and other cells as described in a previous report
(18), reducing Mg-ATP in the internal solution eliminated activation of a store-operated calcium entry current. Because channel rundown is not normally observed over the time course of our
experiments (18), these data indicate that a
phosphorylation event is required for channel activation. This
phosphorylation event is ML-9 sensitive, implicating MLCK in control of
store-operated calcium entry.
The mechanism linking calcium store depletion to activation of a
membrane calcium current remains elusive. Inhibition of store-operated calcium entry by ML-9 implicates involvement of the actin- and myosin-based contractile apparatus in this mechanism. Prior studies (12, 23, 25) have implicated F-actin in control of calcium signaling and, in particular, activation of a store-operated calcium entry current. Indeed, one important role of F-actin may be to maintain
a close physical association between the endoplasmic reticulum and the
cell membrane (23). Cytochalasin D disrupts F-actin and
immediately eliminates endothelial cell tension, increasing the
distance between the endoplasmic reticulum and the plasmalemma. In
voltage-clamped RPAECs, inclusion of cytochalasin D in the patch
pipette activated a calcium current with biophysical properties resembling the calcium current activated by thapsigargin. Stabilizing F-actin with jasplakinolide eliminated the thapsigargin-induced calcium
entry current. Thus our data support the ideas that 1) F-actin conformation is an important determinant of the store-operated calcium entry current and 2) MLCK may control activation of
store-operated calcium entry through stimulation of actomyosin-based
tension. However, our findings are not consistent with recent
observations (6, 22, 25, 40) in other cell types that
F-actin disruption does not influence activation of store-operated
calcium entry. The reasons for these disparate findings are unclear,
although two clear differences between the studies are apparent. In
prior reports, cytochalasin D had not been included in the patch
pipette but, rather, was pretreated. Thus the acute response to
cytochalasin D may differ substantially from its long-term application.
Additionally, prior studies have not utilized endothelial cells.
Although speculative, mechanically sensitive cells like endothelial
cells may possess a greater reliance on cytoskeletal control of
store-operated calcium entry than do other less mechanically sensitive
cell types.
Endothelial cell permeability.
Although an important role for MLCK in control of endothelial
cell barrier function is well established, its mechanism of action is
still incompletely understood (7, 9, 10, 14, 27, 38, 39).
Specifically, it is unclear whether a MLCK-dependent increase in
centripetally directed tension is sufficient to generate intercellular
gaps. Our current data indicate that in addition to stimulating an
inward centripetal tension that pulls cells apart, MLCK could control
calcium entry at the membrane and thus influence signal amplification
through calcium-sensitive targets involved in endothelial cell barrier
function. We therefore performed studies to address the link between
MLCK, calcium entry, and regulation of RPAEC permeability.
Consistent with previous reports (4, 13), activation
of store-operated calcium entry was sufficient to increase endothelial cell permeability. The magnitude of this effect was greatest at 30 min
and decreased in severity over a 2-h time course, indicating that
endothelial cell barrier function improved to near control values.
Prior studies did not assess whether thapsigargin-induced barrier
disruption was reversible; in fact, these data were surprising considering that prolonged exposure to thapsigargin induces cell apoptosis (32). Mechanisms of intercellular gap repair are
poorly understood. Thus it is not presently clear whether repair of the monolayer in our experiments occurred due to resolution of
gap-promoting stimuli, activation of repair mechanisms, or both. A
prior study (26) indicated that
[Ca2+]i stimulation of MLC20
phosphorylation is transient, peaking within 1 min and returning to
baseline levels by 15-30 min. Similarly, calcium stimulation of
phosphatase (PP2b) activity has been implicated in decreasing
MLC20 phosphorylation over prolonged time periods (33). These prior studies suggest that resolution of
gap-promoting stimuli may contribute to resealing barrier function.
However, our studies demonstrated that ML-9 prevented barrier
restoration in the presence of thapsigargin at time points when
MLC20 phosphorylation had returned to baseline levels. Thus
although our data suggest that the resealing of intercellular gaps
proceeds via an ML-9-sensitive mechanism, the link between MLCK,
MLC20 phosphorylation, and actomyosin interaction in
mediating this process is currently unclear.
Reducing [Ca2+]o to 100 nM eliminated
thapsigargin-induced increases in permeability, confirming that barrier
disruption required calcium entry across the cell membrane. These data
are consistent with previous reports linking calcium entry to barrier
disruption (4, 13, 29). Reducing
[Ca2+]o is sufficient to reorganize centrally
localized F-actin while maintaining its peripheral rim
(18). Moreover, in low [Ca2+]o,
thapsigargin neither induces stress fibers nor increases
MLC20 phosphorylation like it does when stimulation of
calcium entry is permitted (18). We have interpreted these
data to suggest that calcium entry is a critical amplification signal
regulating endothelial barrier function.
In our current studies, ML-9 prevented thapsigargin from
stimulating calcium entry but did not prevent thapsigargin from
increasing permeability; rather, inhibition of MLCK activity promoted
the thapsigargin-dependent increase in macromolecular flux. MLCK
inhibition has previously been shown (27) to either
eliminate or partially attenuate permeability induced by Gq
agonists that activate store-operated calcium entry. MLCK inhibition
did not have similar protective effects against the direct
[Ca2+]i-elevating agent ionomycin
(8). In this case, ionomycin disrupted the endothelial
cell barrier by decreasing cAMP content, stimulating tyrosine kinase
activity, and reducing phosphotyrosine incorporation of p125 focal
adhesion kinase. Thapsigargin has previously been shown
(29) to substantially decrease cAMP content, although its
effect on tyrosine kinase activity and phosphotyrosinated substrates
has not been evaluated in this context.
Considering that ML-9 abolished thapsigargin-induced calcium entry, our
data unmask a previously undetermined mechanism of permeability. This
mechanism of barrier regulation was further supported in studies with
low [Ca2+]o where ML-9 alone was sufficient
to induce a large increase in permeability. Thus the data confirm two
distinct mechanisms of barrier disruption: a first mechanism that is
dependent on activation of store-operated calcium entry and a second
mechanism that is exacerbated by low [Ca2+]o
and occurs after ML-9 treatment.
In conclusion, our present studies were predicated around the
idea that endothelial cell tension, established by the function of
MLCK, importantly dictates calcium signaling and endothelial cell
barrier function. Our findings in RPAECs indicate that ML-9 promotes
calcium release from an
Ins(1,4,5)P3 receptor and inhibits activation of store-operated calcium entry channels. Even though activation of store-operated calcium entry is sufficient to increase endothelial cell permeability and inhibition of MLCK prevents calcium
entry, the inhibition of MLCK activity does not prevent thapsigargin
from increasing permeability. Indeed, inhibition of MLCK disrupts
endothelial barrier function, unmasking a novel mechanism regulating
the endothelial cell barrier. Future studies will be required to assess
how the distribution of forces within endothelial cells is altered
after MLCK inhibition, particularly in low
[Ca2+]o, to further address this mechanism of
barrier control.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the assistance of Dr. Paul Babal in
isolation and culture of pulmonary artery endothelial cells. We thank
Judy Creighton and George Brough for excellent technical support.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-56050 and HL-60024 (to T. Stevens) and American Heart
Association Southern Research Consortium Fellowships (to T. M. Moore).
Address for reprint requests and other correspondence: T. Stevens, Dept. of Pharmacology, Univ. of South Alabama, College of
Medicine-MSB 3130, University Blvd., Mobile, AL 36688-0002 (E-mail:
tstevens{at}jaguar1.usouthal.edu).
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. Section 1734 solely to indicate this fact.
Received 19 October 1999; accepted in final form 22 May 2000.
 |
REFERENCES |
1.
Berridge, MJ.
Capacitative calcium entry.
Biochem J
312:
1-11,
1995[ISI][Medline].
2.
Bezprozvanny, I,
and
Ehrlich BE.
The inositol 1,4,5-trisphosphate (InsP3) receptor.
J Membr Biol
145:
205-216,
1995[ISI][Medline].
3.
Bourguignon, LY,
and
Jin H.
Identification of the ankyrin-binding domain of the mouse T-lymphoma cell inositol 1,4,5-trisphosphate (IP3) receptor and its role in the regulation of IP3-mediated internal Ca2+ release.
J Biol Chem
270:
7257-7260,
1995[Abstract/Free Full Text].
4.
Chetham, PM,
Babal P,
Bridges JP,
Moore TM,
and
Stevens T.
Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry.
Am J Physiol Lung Cell Mol Physiol
276:
L41-L50,
1999[Abstract/Free Full Text].
5.
Dean, DA.
Import of plasmid DNA into the nucleus is sequence specific.
Exp Cell Res
230:
293-302,
1997[ISI][Medline].
5a.
Fabiato, A.
Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands.
Methods Enzymol
157:
378-417,
1988[ISI][Medline].
6.
Fagan, KA,
Mons N,
and
Cooper DM.
Dependence of the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells on capacitative Ca2+ entry.
J Biol Chem
273:
9297-9305,
1998[Abstract/Free Full Text].
7.
Garcia, JG,
and
Schaphorst KL.
Regulation of endothelial cell gap formation and paracellular permeability.
J Investig Med
43:
117-126,
1995[ISI][Medline].
8.
Garcia, JG,
Schaphorst KL,
Shi S,
Verin AD,
Hart CM,
Callahan KS,
and
Patterson CE.
Mechanisms of ionomycin-induced endothelial cell barrier dysfunction.
Am J Physiol Lung Cell Mol Physiol
273:
L172-L184,
1997[Abstract/Free Full Text].
9.
Garcia, JG,
Verin AD,
and
Schaphorst KL.
Regulation of thrombin-mediated endothelial cell contraction and permeability.
Semin Thromb Hemost
22:
309-315,
1996[Medline].
10.
Garcia, JG,
Verin AD,
Schaphorst KL,
Siddiqui R,
Patterson CE,
Csortos C,
and
Natarajan V.
Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60src.
Am J Physiol Lung Cell Mol Physiol
276:
L989-L998,
1999[Abstract/Free Full Text].
11.
Gregory, RB,
Wilcox RA,
Berven LA,
van Straten NC,
van der Marel GA,
van Boom JH,
and
Barritt GJ.
Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes.
Biochem J
341:
401-408,
1999[ISI][Medline].
12.
Holda, JR,
and
Blatter LA.
Capacitative calcium entry is inhibited in vascular endothelial cells by disruption of cytoskeletal microfilaments.
FEBS Lett
403:
191-196,
1997[ISI][Medline].
13.
Kelly, JJ,
Moore TM,
Babal P,
Diwan AH,
Stevens T,
and
Thompson WJ.
Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability.
Am J Physiol Lung Cell Mol Physiol
274:
L810-L819,
1998[Abstract/Free Full Text].
14.
Khimenko, PL,
Moore TM,
Wilson PS,
and
Taylor AE.
Role of calmodulin and myosin light-chain kinase in lung ischemia-reperfusion injury.
Am J Physiol Lung Cell Mol Physiol
271:
L121-L125,
1996[Abstract/Free Full Text].
15.
Lum, H,
and
Malik AB.
Regulation of vascular endothelial barrier function.
Am J Physiol Lung Cell Mol Physiol
267:
L223-L241,
1994[Abstract/Free Full Text].
16.
Majno, G,
and
Palade GE.
Studies on inflammation. I. The effect of histamine and serotonin on vascular permeability: an electron microscopic study.
J Biophys Biochem Cytol
11:
571-605,
1961[Abstract/Free Full Text].
17.
Michel, CC,
and
Curry FE.
Microvascular permeability.
Physiol Rev
79:
703-761,
1999[Abstract/Free Full Text].
18.
Moore, TM,
Brough GH,
Babal P,
Kelly JJ,
Li M,
and
Stevens T.
Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.
Am J Physiol Lung Cell Mol Physiol
275:
L574-L582,
1998[Abstract/Free Full Text].
19.
Moy, AB,
Shasby SS,
Scott BD,
and
Shasby DM.
The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells.
J Clin Invest
92:
1198-1206,
1993[ISI][Medline].
20.
Moy, AB,
Van Engelenhoven J,
Bodmer J,
Kamath J,
Keese C,
Giaever I,
Shasby S,
and
Shasby DM.
Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.
J Clin Invest
97:
1020-1027,
1996[Abstract/Free Full Text].
21.
Patel, S,
Morris SA,
Adkins CE,
O'Beirne G,
and
Taylor CW.
Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization.
Proc Natl Acad Sci USA
94:
11627-11632,
1997[Abstract/Free Full Text].
22.
Patterson, RL,
van Rossum DB,
and
Gill DL.
Store-operated Ca2+ entry: evidence for a secretion-like coupling model.
Cell
98:
487-499,
1999[ISI][Medline].
23.
Putney, JW, Jr.
The capacitative model for receptor-activated calcium entry.
Adv Pharmacol
22:
251-269,
1991[Medline].
24.
Putney, JW, Jr,
and
Bird GS.
The signal for capacitative calcium entry.
Cell
75:
199-201,
1993[ISI][Medline].
25.
Ribeiro, CM,
Reece J,
and
Putney JW, Jr.
Role of the cytoskeleton in calcium signaling in NIH 3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+]i signaling, but not for capacitative calcium entry.
J Biol Chem
272:
26555-26561,
1997[Abstract/Free Full Text].
26.
Shasby, DM,
Stevens T,
Ries D,
Moy AB,
Kamath JM,
Kamath AM,
and
Shasby SS.
Thrombin inhibits myosin light chain dephosphorylation in endothelial cells.
Am J Physiol Lung Cell Mol Physiol
272:
L311-L319,
1997[Abstract/Free Full Text].
27.
Sheldon, R,
Moy A,
Lindsley K,
Shasby S,
and
Shasby DM.
Role of myosin light-chain phosphorylation in endothelial cell retraction.
Am J Physiol Lung Cell Mol Physiol
265:
L606-L612,
1993[Abstract/Free Full Text].
28.
Stevens, T,
Fouty B,
Hepler L,
Richardson D,
Brough G,
McMurtry IF,
and
Rodman DM.
Cytosolic Ca2+ and adenylyl cyclase responses in phenotypically distinct pulmonary endothelial cells.
Am J Physiol Lung Cell Mol Physiol
272:
L51-L59,
1997[Abstract/Free Full Text].
29.
Stevens, T,
Nakahashi Y,
Cornfield DN,
McMurtry IF,
Cooper DM,
and
Rodman DM.
Ca(2+)-inhibitable adenylyl cyclase modulates pulmonary artery endothelial cell cAMP content and barrier function.
Proc Natl Acad Sci USA
92:
2696-2700,
1995[Abstract].
30.
Taylor, CW,
and
Broad LM.
Pharmacological analysis of intracellular Ca2+ signalling: problems and pitfalls.
Trends Pharmacol Sci
19:
370-375,
1998[ISI][Medline].
31.
Thastrup, O,
Cullen PJ,
Drobak BK,
Hanley MR,
and
Dawson AP.
Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.
Proc Natl Acad Sci USA
87:
2466-2470,
1990[Abstract].
32.
Tsukamoto, A,
and
Kaneko Y.
Thapsigargin, a Ca(2+)-ATPase inhibitor, depletes the intracellular Ca2+ pool and induces apoptosis in human hepatoma cells.
Cell Biol Int
17:
969-970,
1993[ISI][Medline].
33.
Verin, AD,
Cooke C,
Herenyiova M,
Patterson CE,
and
Garcia JG.
Role of Ca2+/calmodulin-dependent phosphatase 2B in thrombin-induced endothelial cell contractile responses.
Am J Physiol Lung Cell Mol Physiol
275:
L788-L799,
1998[Abstract/Free Full Text].
34.
Verin, AD,
Gilbert-McClain LI,
Patterson CE,
and
Garcia JG.
Biochemical regulation of the nonmuscle myosin light chain kinase isoform in bovine endothelium.
Am J Respir Cell Mol Biol
19:
767-776,
1998[Abstract/Free Full Text].
35.
Verin, AD,
Lazar V,
Torry RJ,
Labarrere CA,
Patterson CE,
and
Garcia JG.
Expression of a novel high molecular-weight myosin light chain kinase in endothelium.
Am J Respir Cell Mol Biol
19:
758-766,
1998[Abstract/Free Full Text].
36.
Watanabe, H,
Takahashi R,
Zhang XX,
Goto Y,
Hayashi H,
Ando J,
Isshiki M,
Seto M,
Hidaka H,
Niki I,
and
Ohno R.
An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells.
FASEB J
12:
341-348,
1998[Abstract/Free Full Text].
37.
Watanabe, H,
Takahashi R,
Zhang XX,
Kakizawa H,
Hayashi H,
and
Ohno R.
Inhibition of agonist-induced Ca2+ entry in endothelial cells by myosin light-chain kinase inhibitor.
Biochem Biophys Res Commun
225:
777-784,
1996[ISI][Medline].
38.
Wysolmerski, RB,
and
Lagunoff D.
Involvement of myosin light-chain kinase in endothelial cell retraction.
Proc Natl Acad Sci USA
87:
16-20,
1990[Abstract].
39.
Wysolmerski, RB,
and
Lagunoff D.
Regulation of permeabilized endothelial cell retraction by myosin phosphorylation.
Am J Physiol Cell Physiol
261:
C32-C40,
1991[Abstract/Free Full Text].
40.
Yao, Y,
Ferrer-Montiel AV,
Montal M,
and
Tsien RY.
Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger.
Cell
98:
475-485,
1999[ISI][Medline].
Am J Physiol Lung Cell Mol Physiol 279(5):L815-L824
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