Increased expression of calreticulin is linked to ANG
IV-mediated activation of lung endothelial NOS
Jawaharlal M.
Patel1,2,
Yong D.
Li2,
Jianliang
Zhang2,
Craig H.
Gelband3,
Mohan K.
Raizada3, and
Edward R.
Block1,2
1 Research Service, Malcom
Randall Department of Veterans Affairs Medical Center, and
Departments of 2 Medicine and
3 Physiology, University of
Florida College of Medicine, Gainesville, Florida 32608
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ABSTRACT |
This study
demonstrates that ANG IV-induced activation of lung endothelial cell
nitric oxide synthase (ecNOS) is mediated through mobilization of
Ca2+ concentration and by
increased expression and release of the Ca2+ binding protein calreticulin
in pulmonary artery endothelial cells (PAEC). In
Ca2+-free medium and in the
presence of the ANG II AT1 and
AT2 receptor antagonists losartan
and PD-123319 (1 µM each), respectively, ANG IV (5, 50, and 500 nM)
significantly increased intracellular Ca2+ release in PAEC
(P < 0.05 for all concentrations).
In contrast, ANG IV-mediated activation of ecNOS was abolished by the
intracellular Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. ANG IV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG IV stimulation (P < 0.05). Catalytic activity of
purified ecNOS in the absence of calmodulin was increased in a
concentration-dependent fashion by calreticulin. Immunocoprecipitation
studies revealed that ecNOS and calreticulin were coprecipitated in ANG
IV-stimulated PAEC. These results demonstrate that ANG IV-mediated
activation of ecNOS is regulated by intracellular
Ca2+ mobilization and by increased
expression of calreticulin, which appears to involve interaction of
ecNOS and calreticulin proteins in PAEC.
endothelial cell nitric oxide synthase; calcium; angiotensin IV; protein interaction; protein synthesis; protein-protein interaction
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INTRODUCTION |
WE RECENTLY REPORTED that angiotensin IV (ANG IV)
activates the constitutively expressed lung endothelial cell isoform of nitric oxide synthase (ecNOS) by a receptor-mediated pathway, leading
to increases in nitric oxide (NO) release, production of cGMP, and
NO-cGMP-mediated porcine pulmonary arterial vasodilation (14, 31). The
catalytic activity of ecNOS is
Ca2+ and calmodulin dependent and
is transiently activated by agonist-mediated signaling pathways that
increase mobilization of intracellular Ca2+ (7, 11, 15). These signaling
mechanisms are associated with activation of several
Ca2+-dependent enzymes responsible
for mediating vascular endothelial cell function (3, 7, 16).
Agonist-mediated intracellular Ca2+ mobilization also plays a
critical role as a second messenger in the regulation of a variety of
cell functions, including protein expression, cell proliferation, gene
expression, and protein-protein interaction (4, 10, 17, 23, 25).
Depletion of Ca2+ from the
endoplasmic reticulum (ER) or sarcoplasmic reticulum, a known
intracellular Ca2+ storage site,
can facilitate a process that results in upregulation of a group of
Ca2+ binding proteins, including
calreticulin, located within the lumen of the ER (6, 22, 27).
Calreticulin is a 60-kDa, ubiquitous
Ca2+ binding protein of the ER
that consists of low- and high-affinity binding sites and is localized
in various subcellular compartments, including the cytosol, the
nucleus, and the cell surface membrane (1, 24, 37). Calreticulin has
been reported to be secreted from cells (4, 28) and is present at low
levels in human plasma (35). Calreticulin has also been recognized as a
multifunctional protein involved in a wide variety of cellular
processes, including its interaction with endothelium in canine
coronary arteries, which results in stimulated NO production (9, 18).
However, the mechanism by which calreticulin increases NO production by
vascular endothelium is unknown. Because ANG IV-stimulated activation
of ecNOS and release of NO are mediated by a posttranscriptional mechanism and because catalytic activity of ecNOS is elevated from 0.5 to 12 h after ANG IV stimulation (31), we examined whether ANG
IV-mediated early and sustained activation of ecNOS is regulated
through 1) intracellular
Ca2+ release and
2) increased expression and release
of calreticulin involving an ecNOS-calreticulin interaction in lung
endothelial cells.
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MATERIALS AND METHODS |
Cell culture and treatment.
Endothelial cells were isolated from the main pulmonary artery of 6- to
7-mo-old pigs and propagated in monolayers, as previously described
(30). Third- to fourth-passage cells in postconfluent monolayers
maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY)
with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) were used
in all experiments. In each experiment, cell monolayers were studied 1 or 2 days after confluence and were matched for cell line, passage, and
days after confluence.
To determine the potential effect of ANG IV on
Ca2+ release from intracellular
stores, cell monolayers in 35-mm culture dishes containing a round
coverslip were incubated with 5 µM membrane-permeant fura 2-AM (fura
2-AM dissolved in 1 mM DMSO stock solution) in Tyrode solution
(composition in mM: 134 NaCl, 5.4 KCl, 2.0 MgCl2, 0.3 NaH2PO4,
10.0 HEPES, and 10.0 dextrose, pH 7.4) for 30 min at 37°C and then
washed to remove excess external fura 2-AM. The fura 2-loaded cells
were then incubated (1 min) in the presence of 1 µM each losartan (an
ANG II AT1-receptor antagonist)
and PD-123319 (an ANG II
AT2-receptor antagonist) and
stimulated with ANG IV (5, 50, or 500 nM), and intracellular
Ca2+ concentration
([Ca2+]i)
was measured (see below).
To examine the role of Ca2+ in ANG
IV-stimulated activation of ecNOS, cell monolayers were preincubated in
Tyrode solution with or without 2 mM
CaCl2 with and without 50 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (dissolved in 1 mM DMSO stock solution) for 30 min at
37°C, then incubated 2 h at 37°C with or without 1 µM ANG IV.
Controls were incubated in Tyrode solution alone under identical conditions. For studies involving an extracellular
Ca2+-free environment, the Tyrode
solution was supplemented with 5 mM EGTA. After incubation, the cells
were used to measure total membrane fraction ecNOS activity.
To examine the effect of ANG IV on calreticulin expression and release
of calreticulin into the medium, cell monolayers were incubated in RPMI
1640 with or without ANG IV (1 µM) for 2-12 h at 37°C. After
incubation, calreticulin levels in the cells were determined by
two-dimensional gel electrophoresis, microsequencing, and Western blot
analysis. For estimation of released calreticulin in the medium, an
equal volume (60 ml each) of medium from control cells and from cells
incubated with ANG IV for 2 h was concentrated to 5 ml with use of a
Centriprep-30 concentrator (Amicon, Beverly, MA) and analyzed by
Western blot. To examine whether ANG IV-induced intracellular
Ca2+ release is required for
increased calreticulin expression, in some experiments, cell monolayers
were preincubated in Tyrode solution without
Ca2+ with or without 50 µM
BAPTA-AM for 30 min at 37°C, then incubated for 2, 4, and 6 h at
37°C with or without 1 µM ANG IV. After incubation, calreticulin
levels in the cell lysates were determined by Western blot analysis.
[Ca2+]i
measurement.
[Ca2+]i
was measured using epifluorescence microscopy (36). Briefly, the fura
2-AM-loaded cells were alternately illuminated with ultraviolet light
at 340- and 380-nm wavelengths with use of an IonOptix (Milton, MA)
electronically controlled dual-excitation imaging fluorescence system.
Cell fluorescence (emitted light) was collected through a 510-nm
barrier filter before acquisition by a photomultiplier tube. The
fluorescence signals at 340 and 380 nm
(F340 and
F380, respectively) were
background subtracted, i.e., fluorescence signal from fura 2-AM-loaded
ANG IV-unstimulated cells, during the experiment. The mean changes in
F340 to F380 ratios were graphed to give a
relative indication of the changes observed in
[Ca2+]i. The percent intracellular
Ca2+ release was determined from the F340 to
F380 ratio (36).
Measurement of ecNOS activity.
ecNOS activity was measured by monitoring the formation of
L-[3H]citrulline
from
L-[3H]arginine
in the total membrane fraction (31, 32). Total membranes (100-200
µg of protein) were incubated (total volume 0.4 ml) in buffer (50 mM
Tris · HCl, 0.1 mM each EDTA and EGTA, 1 mM
phenylmethylsulfonyl fluoride, and 1 mg/l leupeptin, pH 7.4) containing
1 mM NADPH, 100 nM calmodulin, 10 µM tetrahydrobiopterin, and 5 µM combined L-arginine and
purified
L-[3H]arginine
for 30 min at 37°C. Purification of
L-[3H]arginine
and measurement of L-citrulline
formation were carried out as previously described (29).
Two-dimensional gel electrophoresis and identification of
calreticulin.
Two-dimensional electrophoresis of control and ANG IV-stimulated cells
was performed according to the method of O'Farrell (26) with use of a
Bio-Rad protein II xi system (Bio-Rad, Richmond, CA), as previously
described (20). Briefly, in the first dimension, the isoelectric
focusing gel was loaded with equal amounts (180 µg) of
TCA-precipitable cell lysate proteins. The second dimension (SDS-PAGE)
was performed on a 7.5% separating Laemmli gel with a 3.9% stacking
gel (20).
To identify calreticulin, immunoblot analyses of the two-dimensional
polyacrylamide gels of the cell lysate proteins as well as of gels
loaded with aliquots of concentrated (50 µl) medium from control and
ANG IV-stimulated cells were performed. Proteins were
electrophoretically transferred from the slab gels to polyvinylidine difluoride membranes, as described previously (20). To saturate nonspecific binding sites, the membranes were blocked by 1%
blot-qualified BSA (Promega, Madison, WI) in 20 mM
Tris · HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 for 1 h. The immunodetection was performed with a monoclonal human
anti-calreticulin antibody and an anti-rabbit IgG horseradish
peroxidase-linked whole antibody (Upstate Biotechnology, Lake Placid,
NY). Immunoreactivity was detected by enhanced chemiluminescence (Amersham) (20). The blots were scanned using the Fluor-S MultImager system (Bio-Rad) to quantify protein content.
Expression and purification of ecNOS.
Escherichia coli transformed with the
plasmid Bov-eNOSpCW (kindly provided by Dr. B. S. S. Masters,
University of Texas, San Antonio, TX) was incubated in 0.5 liter of
modified TB (20 g yeast extract, 10 g bactotryptone, 2.65 g
KH2PO4,
4.33 g
Na2HPO4,
and 4 ml glycerol per liter) containing ampicillin (50 µg/ml) and chloramphenicol (35 µg/ml). The cultures were grown in an orbital shaker (20 rpm; Forma Scientific) in the presence of 0.5 mM
-aminolevulinic acid at 22°C. After 1 h of incubation,
ecNOS gene expression was induced by
adding 0.5 mM isopropyl
-D-thiogalactopyranoside, 3 µM riboflavin, and 1 mM ATP. The flasks were kept on an orbital shaker in the dark at 22°C (200 rpm) for 48 h. After incubation, the cultures were centrifuged at 5,000 rpm for 10 min at 5°C, and
the cell pellets were collected and used for purification of ecNOS.
Purification of ecNOS was carried out essentially as described
previously with use of 2',5'-ADP-Sepharose 4B affinity
chromatography (32). Briefly, the cell pellets were resuspended in
buffer A (50 mM
Tris · HCl, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 150 mM NaCl, 10% glycerol, and leupeptin and pepstatin at 1 µM each), lysed by pulsed sonication, and centrifuged at 100,000 g for 30 min at 4°C. The supernatants were applied to
2',5'-ADP-Sepharose 4B (Pharmacia Biotech) columns equilibrated in buffer B (50 mM
Tris · HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM
dithiothreitol, 150 mM NaCl, and 10% glycerol). The columns were
washed with 10 column volumes of buffer
B, then with 10 column volumes of
buffer B containing 600 mM NaCl, and
then they were eluted with buffer B
containing 600 mM NaCl and 5 mM adenosine 2'-monophosphate. The
purity of the enzyme was determined by monitoring ecNOS activity as
well as by Western blot analysis (39).
Calreticulin and catalytic activity of purified ecNOS.
To determine whether the catalytic activity of ecNOS is supported by
calreticulin in the absence of calmodulin, purified ecNOS (3 µg
protein) was incubated with increasing amounts of calreticulin (10-70 nM) for 10 min at 37°C. After incubation, the catalytic activity of ecNOS was monitored as described in
Measurement of ecNOS activity, except
for the absence of calmodulin in the incubation mixture (31, 32). In
some experiments, purified ecNOS (3 µg protein) was incubated with 50 nM calreticulin for 10 min at 37°C, and then the catalytic activity
of ecNOS was monitored in the absence of calmodulin but in the presence
of increasing concentrations (10-1,000 nM) of
Ca2+. To determine the effects of
the combination of calreticulin and calmodulin, purified ecNOS (3 µg
of protein in each sample) was incubated as described in
Measurement of ecNOS activity, except 100 nM calmodulin in the reaction mixture was replaced with various ratios of calreticulin to calmodulin (60:10-10:60 nM), with 60 nM
calreticulin alone, or with 60 nM calmodulin alone. After 30 min of
incubation at 37°C, the catalytic activity of ecNOS was measured by
monitoring the formation of
L-[3H]citrulline
from
L-[3H]argnine
(31, 32).
Immunoprecipitation and analysis of ecNOS and calreticulin.
Cell monolayers stimulated with or without ANG IV (1 µM) at 37°C
for 4 h were lysed in buffer consisting of 20 mM
Tris · HCl, pH 7.4, 2.5 mM EDTA, 100 mM NaCl, 10 mM
sodium fluoride, 1 mM sodium vanadate, 1 mM Pefabloc, 1% Triton X-100,
1% sodium deoxycholate, 0.1% SDS, and 10 µg/ml each of pepstatin
and leupeptin. The cell lysate proteins (50 µg) or buffer only
(blanks) was incubated with 1 µg of anti-ecNOS monoclonal antibody
(Transduction Laboratories, Lexington, KY) for 1 h at 4°C, and then
20 µl of Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Santa
Cruz, CA) were added and incubated with mixing overnight at 4°C.
The reaction mixtures were centrifuged (2,500 rpm) for 15 min at
4°C, and the agarose pellets were collected, washed with PBS
containing 1 M NaCl three times, and then boiled in 40 µl of loading
buffer for 90 s. The samples were fractionated on a 7.5% SDS
polyacrylamide gel and blotted onto polyvinylidine difluoride membranes
(39). The blots were hybridized with monoclonal anti-ecNOS and
anti-calreticulin antibodies, and the immunoreactive bands were
visualized by enhanced chemiluminescence detection (39).
Statistical analysis.
Statistical significance for the effect of ANG IV and BAPTA-AM on ecNOS
activity, intracellular Ca2+
release, and calreticulin expression and for the effect of calreticulin on ecNOS activity was determined using ANOVA and Student's paired t-test (38). Values are means ± SE
for n experiments.
P < 0.05 was taken as significant.
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RESULTS |
ANG IV stimulation increases
[Ca2+]i.
ANG IV stimulated an increase in
[Ca2+]i
in pulmonary artery endothelial cells (PAEC) as illustrated in Fig.
1. ANG IV caused rapid increases in
[Ca2+]i
in a dose-dependent manner, and the increases were significantly greater than the basal level of
[Ca2+]i
(P < 0.05 for all concentrations).

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Fig. 1.
ANG IV increases intracellular
Ca2+
(Ca2+i) release in a dose-dependent
manner. Pulmonary arterial endothelial cell (PAEC) monolayers loaded
with fura 2-AM were preincubated in Tyrode solution without
Ca2+ in presence of losartan and
PD-123319 (1 µM each), then stimulated with 5, 50, and 500 nM ANG IV.
Left: ANG IV concentration-dependent
increases in fluorescence ratio [ratio of fluorescence at 340 nm
to that at 380 nm
(F340/F380)],
representing transient intracellular
Ca2+ release.
Right: percent intracellular
Ca2+ release from 4 independent
samples. * P < 0.001 vs. basal
level in presence of losartan and PD-123319.
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ANG IV-mediated intracellular
Ca2+ release is
associated with ecNOS activation.
To examine whether ANG IV-induced intracellular
Ca2+ release is associated with
activation of ecNOS, we examined the effect of the intracellular
Ca2+ chelator BAPTA-AM on ecNOS
activity. As shown in Fig. 2, incubation of
PAEC in the presence of ANG IV but in the absence of extracellular Ca2+ significantly
(P < 0.05) increased ecNOS activity
compared with control. In contrast, preincubation of cell monolayers
with BAPTA-AM in the absence of extracellular
Ca2+ completely blocked ANG
IV-stimulated activation of ecNOS. ecNOS activity was comparable to
controls when cells were incubated with BAPTA-AM alone under identical
conditions. Similar results were obtained if cell monolayers were
stimulated by ANG IV in the presence of extracellular
Ca2+ (data not shown).

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Fig. 2.
Effect of intracellular Ca2+
chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM on ANG IV-stimulated endothelial cell nitric oxide
synthase (ecNOS) activation. Porcine PAEC were incubated in Tyrode
solution without Ca2+ with or
without 50 µM BAPTA-AM for 30 min, then incubated for 2 h at 37°C
with or without 1 µM ANG IV. Respective controls were incubated in
Tyrode solution alone or in Tyrode solution containing 50 µM BAPTA-AM
under identical conditions. After incubation, total membrane fraction
ecNOS activity was measured. Similar results were obtained when cell
monolayers were incubated in RPMI 1640 (data not shown). Values are
means ± SE (n = 4).
* P < 0.05 vs. control.
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ANG IV increases expression and release of calreticulin.
Immunoblot analysis of two-dimensional gels of control and ANG
IV-stimulated cells is shown in Fig. 3. A
human monoclonal antibody for calreticulin reacted with a 60-kDa
protein, and the intensity of this reaction was increased severalfold
in cells exposed to ANG IV for 12 h compared with controls. To confirm the identity of calreticulin,
NH2-terminal amino acid sequence analysis of the 60-kDa protein followed by protein database analysis of
25 residues (EPTIYFKEQFLDGDGWTDRWIESKH) matched 100% with rabbit uterine calreticulin and 96% with human placental calreticulin. The
time course of the ANG IV-stimulated increased expression of
calreticulin was determined and is shown in Fig.
4. ANG IV-mediated expression of
calreticulin was significantly increased as early as 2 h and remained
elevated for 6 h (P < 0.05 for all
time points). In addition to increased expression of cellular
calreticulin, similar immunoreactivity and increased levels of
calreticulin were observed in medium from cells stimulated with ANG IV
compared with medium from control cells (Fig.
5).

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Fig. 3.
Western blot analysis of calreticulin after ANG IV stimulation. PAEC
monolayers in RPMI 1640 containing 1 µM ANG IV
(B) or RPMI 1640 only (control,
A) were incubated for 12 h at 37°C. Cell
lysate proteins (180 µg) were separated by 2-dimensional
electrophoresis and immunoanalyzed. pI, isoelectric point.
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Fig. 4.
Time-dependent effect of ANG IV on calreticulin expression. PAEC
monolayers in RPMI 1640 containing 1 µM ANG IV or RPMI 1640 alone
(Con) were incubated for 2, 4, and 6 h at 37°C. Cell lysate
proteins (50 µg) were fractionated on a 7.5% SDS polyacrylamide gel,
blotted onto polyvinylidine difluoride (PVDF) membranes and then
hybridized with human anti-calreticulin monoclonal antibody. Blots were
analyzed by densitometric analysis to quantify calreticulin protein
content. A: representative data from 1 of 4 independent experiments. MW, molecular mass.
B: results of densitometric analysis
of blots from 4 independent experiments (means ± SE).
* P < 0.05 vs. control
(Con).
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Fig. 5.
ANG IV-stimulated expression of calreticulin increases its secretion
into extracellular medium. PAEC monolayers in RPMI 1640 containing 1 µM ANG IV or RPMI 1640 only (Con) were incubated for 2 h at 37°C.
After incubation, 60 ml of media were collected and concentrated, and
calreticulin was identified by immunoblot analysis.
A: representative data from 1 of 3 independent experiments. B: results of densitometric
analysis of blots from 3 independent experiments (means ± SE).
* P < 0.05 vs. control.
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ANG IV-mediated level of intracellular
Ca2+ release is
critical for expression of calreticulin.
To determine whether ANG IV-induced
[Ca2+]i
is critical for increased expression of calreticulin, we examined the
effect of the intracellular Ca2+
chelator BAPTA-AM on calreticulin expression. As shown in Fig. 6, ANG IV-induced expression of
calreticulin was blocked by BAPTA-AM.

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Fig. 6.
Effect of intracellular Ca2+
chelator on ANG IV-mediated calreticulin expression. PAEC monolayers
were incubated in Tyrode solution without
Ca2+ with or without 50 µM
BAPTA-AM for 30 min, then incubated for 2, 4, or 6 h at 37°C with
or without 1 µM ANG IV. After incubation, cell lysate proteins (50 µg) were fractionated on a 7.5% SDS polyacrylamide gel, blotted onto
PVDF membranes, and then hydridized with human anti-calreticulin
monoclonal antibody.
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Calreticulin increases catalytic activity of ecNOS in the absence of
calmodulin.
Because the Ca2+ binding
protein calmodulin is critical for the catalytic activity of ecNOS, we
determined whether substitution of calreticulin for calmodulin can
maintain catalytic activity of ecNOS. As shown in Fig.
7A, in the
absence of calmodulin, calreticulin increased the catalytic activity of
purified ecNOS in a dose-dependent manner. The effect of various
concentrations of Ca2+ on
calreticulin-mediated activation of ecNOS revealed that calreticulin can increase catalytic activity at physiologically relevant
concentrations of Ca2+ (Fig.
7B).

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Fig. 7.
Calreticulin and Ca2+
concentration-dependent effects on catalytic activity of purified ecNOS
in absence of calmodulin. Purified ecNOS (3 µg of protein) was
incubated in presence of increasing concentrations (10-70 nM) of
calreticulin for 10 min at 37°C
(A) or in presence of 50 nM
calreticulin and increasing concentrations (10-1,000 nM) of
Ca2+ for 10 min at 37°C
(B). After incubation, catalytic
activity of ecNOS was measured in absence of calmodulin but in presence
of other cofactors. Values are means ± SE;
n = 3 for each data point.
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Calreticulin-to-calmodulin ratio is critical for increased catalytic
activity of ecNOS.
To identify possible competitive effects between calreticulin and
calmodulin on ecNOS activity, the catalytic activity of purified ecNOS
was determined in the presence of increasing or decreasing
concentrations of calmodulin and calreticulin. As shown in Fig.
8A, in the
absence of calreticulin, catalytic activity of ecNOS was increased by
calmodulin in a dose-dependent manner, with maximal activation observed
at 50 nM calmodulin. In the absence of calmodulin, calreticulin (60 nM)
caused only a limited increase in the catalytic activity of ecNOS (Fig.
8B). However,
calreticulin-to-calmodulin ratios of 60:10, 50:20, 40:30, and 30:40
significantly increased the catalytic activity of ecNOS compared with
ecNOS activities at 10, 20, 30, and 40 nM calmodulin alone
(P < 0.05 for all; Fig. 8B), indicating that calreticulin
can enhance the catalytic activity of ecNOS in the presence of
calmodulin. Calreticulin or calmodulin alone at 60 nM increased the
catalytic activity of ecNOS, but the calmodulin-mediated activation of
ecNOS was severalfold greater than that observed with 60 nM
calreticulin alone (Fig. 8B).
Western blot analysis revealed that endogenous calmodulin levels in
cells incubated for 2-12 h with ANG IV were comparable to controls
(data not shown).

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Fig. 8.
Effect of calreticulin-to-calmodulin ratio on catalytic activity of
ecNOS. A: purified ecNOS was incubated
in absence of calreticulin but in presence of increasing concentrations
(10-70 nM) of calmodulin. B:
purified ecNOS was incubated with decreasing (60-10 nM)
calreticulin and increasing (10-60 nM) calmodulin concentrations,
with 60 nM calreticulin alone, or with 60 nM calmodulin alone for 10 min at 37°C. After incubation, catalytic activity of ecNOS was
monitored. Values are means ± SE;
n = 6 for each data point.
* P < 0.05 vs. 10-40 nM
calmodulin alone.
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Calreticulin interacts with ecNOS protein.
To determine whether calreticulin directly interacts with
ecNOS in intact PAEC, ecNOS from control and ANG
IV-stimulated cells was immunoprecipitated using anti-ecNOS monoclonal
antibody, and the immunoprecipitates were analyzed for the presence of
ecNOS and calreticulin proteins. As shown in Fig.
9, the presence of ecNOS and calreticulin
proteins in the immunoprecipitates suggests that ecNOS exists in a
complex with calreticulin in PAEC.

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Fig. 9.
Interaction between ecNOS and calreticulin. PAEC monolayers in RPMI
1640 containing 1 µM ANG IV or RPMI 1640 alone (control, C) were
incubated for 4 h at 37°C. After incubation, cell lysate proteins
(50 µg) or buffer only (blank, B) was immnoprecipitated with
anti-ecNOS monoclonal antibody. Immunoprecipitated complex was
denatured by boiling, fractionated, blotted on PVDF membranes, and
hybridized with monoclonal ecNOS and calreticulin antibodies. Data are
representative of 2 independent experiments with similar results.
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 |
DISCUSSION |
We demonstrate here that ANG IV stimulation in the presence of ANG II
AT1 and
AT2 antagonists increases
[Ca2+]i,
which is associated with increased catalytic activity of ecNOS in PAEC.
Our results further demonstrate for the first time that ANG IV
stimulation results in increased expression and release of the
Ca2+ binding protein calreticulin.
The ANG IV-mediated activation of ecNOS and the ANG IV-mediated
increase in expression of calreticulin are linked to release of
intracellular Ca2+, and these
responses were completely diminished by the intracellular Ca2+ chelator BAPTA-AM. The
results of the present study are consistent with the well-established
mechanism that agonist-induced transient release of intracellular
Ca2+ can increase catalytic
activity of ecNOS (7, 11, 15). However, ANG IV-mediated increased
expression of calreticulin and its association with a sustained
increase in ecNOS activity are of physiological relevance, because we
previously reported that ANG IV-mediated increased catalytic activity
of ecNOS is directly linked to increased NO production and pulmonary
artery vasorelaxation through the NO-cGMP signaling mechanism (14, 31).
ANG IV-mediated activation of ecNOS and increased expression of
calreticulin are clearly dependent on intracellular
Ca2+ release, inasmuch as these
responses were abrogated by the intracellular Ca2+ chelator BAPTA-AM.
Agonist-mediated cytosolic Ca2+
release is associated with receptor-linked signaling pathways. One of
the most ubiquitous pathways is activation of the phospholipase C-inositol 1,4,5-trisphosphate pathway, which releases
Ca2+ from intracellular
Ca2+ pools, namely, the ER (2).
Although the precise signaling pathway involved in ANG IV-mediated
intracellular Ca2+ release remains
to be determined, two pieces of evidence indicate that the ER is the
most likely source of the cytosolic
Ca2+ elevation. First, ANG
IV-stimulated increased intracellular
Ca2+ release and activation of
ecNOS were observed in the absence or presence of extracellular
Ca2+, suggesting that elevation of
cytosolic Ca2+ and activation of
ecNOS were due to its release from intracellular stores and not as a
result of increased influx. Second, our results demonstrated that the
ANG IV-mediated increased expression of calreticulin was blocked by the
intracellular Ca2+ chelator
BAPTA-AM, and this is consistent with reports demonstrating an
association between the depletion of ER
Ca2+ stores and increased
expression of a set of ER resident proteins, including calreticulin, in
a variety of cells (5, 16, 27).
Our results also demonstrate that ANG IV-mediated increased expression
of calreticulin resulted in release of this protein into the medium.
Calreticulin secretion and the presence of calreticulin in human plasma
have been previously reported (4, 35). Although the mechanism of
secretion of calreticulin is unknown, it is suggested that perturbation
of cellular Ca2+ enhances
secretion of luminal ER proteins including calreticulin (4). The ANG
IV-induced increased expression and secretion of calreticulin is
particularly important, because a recent study by Kuwabara et al. (18)
demonstrated that infusion of calreticulin increases endothelial cell
surface binding in canine coronary artery and stimulates NO production.
Although the mechanism of calreticulin-mediated NO generation was not
examined by these authors, our results demonstrate that incubation of
purified ecNOS with calreticulin increases catalytic activity of ecNOS,
suggesting that the increased NO production is most likely associated
with increased catalytic activity of ecNOS. Further examination of calreticulin-mediated activation of ecNOS revealed that, in the absence
of calmodulin, calreticulin increases catalytic activity in a
concentration-dependent manner. This calreticulin-mediated increase in
ecNOS activity occurred in the presence of physiologically relevant
concentrations of Ca2+. In
addition, calreticulin enhanced the catalytic activity of ecNOS in the
presence of calmodulin in vitro. We believe that a similar situation
exists in cells stimulated with ANG IV, because calreticulin content
was increased in these cells, but endogenous calmodulin expression was
not changed.
Finally, our results suggest a protein-protein interaction between
ecNOS and calreticulin in porcine PAEC. The existence of such a complex
would provide a cellular construct for understanding how ANG
IV-mediated increases in calreticulin result in ecNOS activation.
Although the precise nature of the interaction between ecNOS and
calreticulin remains to be determined, it is possible that the
Ca2+ binding site of ecNOS protein
may be conformationally changed by calreticulin. If this is so, it
appears that such a molecular interaction might enhance the effect of
calmodulin on the catalytic activity of ecNOS. Alternatively, an
increased level of calreticulin may enhance the potential interactions
between ecNOS and other proteins, such as heat shock protein 90, reported to be involved in ecNOS activation (12). This possibility is
supported by reports that calreticulin can interact with other proteins
such as nuclear hormone receptors, integrin
-subunit, and steroid
hormone receptors, resulting in modulation of their function (5, 8,
19).
The physiological significance of the observations reported here
relates not only to ANG IV-mediated sustained activation of ecNOS and
vascular regulation through the NO-cGMP/signaling mechanism but to the
multifunctional nature of calreticulin. For example, calreticulin has
been shown to reduce intimal hyperplasia after arterial injury in the
rat (9) and to protect lung and other tissues from a variety of
pathophysiological conditions, including oxidant injury and heat shock
(6, 21). Local release of calreticulin may alter
Ca2+ homeostasis and generate
excessive NO production, resulting in activation of inflammatory
responses in the vasculature. In addition, vasostatin, a calreticulin
fragment, has been reported to inhibit angiogenesis and to suppress
tumor growth in mice (33). Similarly, NO was reported to play a role in
suppression of angiogenesis and endothelial cell migration (33, 34).
Our results emphasize the potential role of ANG
IV/calreticulin-mediated responses in regulation of vascular function.
 |
ACKNOWLEDGEMENTS |
We thank Bert Herrera for tissue culture assistance, Janet Wootten
for excellent editorial help, Addy Heimer for secretarial assistance,
and Weihong Han and Di-hau He for technical assistance.
 |
FOOTNOTES |
This work was supported by the Medical Research Service of the
Department of Veterans Affairs and by National Heart, Lung, and Blood
Institute Grant HL-58679.
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.
Address for reprint requests and other correspondence: J. M. Patel,
Research Service (151), VA Medical Center, 1601 SW Archer Rd.,
Gainesville, FL 32608-1197 (E-mail:
Pateljm{at}medicine.ufl.edu).
Received 30 March 1999; accepted in final form 14 May 1999.
 |
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