Role of calpain in hypoxic inhibition of nitric oxide
synthase activity in pulmonary endothelial cells
Yunchao
Su1 and
Edward R.
Block1,2
1 Department of Medicine, University of
Florida College of Medicine, and 2 Research
Service, Malcom Randall Department of Veterans Affairs Medical
Center, Gainesville, Florida 32608-1197
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ABSTRACT |
Pulmonary artery endothelial cells (PAEC) were exposed to
normoxia or hypoxia (0% O2-95% N2-5%
CO2) in the presence and absence of calpain inhibitor I or
calpeptin, after which endothelial nitric oxide synthase (eNOS)
activity and protein content were assayed. Exposure to hypoxia
decreased eNOS activity but not eNOS protein content. Both calpain
inhibitor I and calpeptin prevented the hypoxic decrease of eNOS
activity. Incubation of calpain with total membrane preparations of
PAEC caused dose-dependent decreases in eNOS activity independent of
changes in eNOS protein content. Exposure of PAEC to hypoxia also
caused time-dependent decreases of heat shock protein 90 (HSP90) that
were prevented by calpain inhibitor I and calpeptin. Moreover, the
HSP90 content in anti-eNOS antibody-induced immunoprecipitates from
hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the
decrease of eNOS activity in these immunoprecipitates. Incubation of
PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the
hypoxic decrease of eNOS activity. These results indicate that the
hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease
in HSP90 caused by calpain activation.
hypoxia; heat shock protein 90; pulmonary artery
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INTRODUCTION |
CALPAIN IS THE NAME USED to describe a family of
calcium-activated, nonlysosomal neutral cysteine endopeptidases that
are ubiquitously distributed in all mammalian cells (15, 17, 21). For
the most part, calpain isoforms consist of a distinct larger catalytic
subunit (about 80 kDa) and a common smaller subunit (about 30 kDa) that
help regulate their activity (10, 21). The cDNA of the subunits has
thus far been cloned in a variety of tissues, including endothelial
cells (6, 10, 29). Calpain exists in the cytosol as procalpain, an
inactive proenzyme that translocates from the cytosol to the cell
membrane in the presence of calcium. Autocatalytic activation of
procalpain to active calpain occurs at the membrane in the presence of
physiological levels of calcium and phosphatidylinositol (4).
Substrates for calpain include the cytoskeletal proteins fodrin, talin,
and filamin; microtubule-associated proteins; and a number of membrane
proteins, including growth factor receptors (e.g., epidermal growth
factor receptor), adhesion molecules (e.g., integrin, cadherin), and ion transporters (e.g., Ca2+-ATPase), as well as enzymes
such as protein kinase C, myosin light chain kinase,
calmodulin-dependent kinase, phosphatases, and phospholipase C (21).
Calpain has been found to have an important role in transmembrane
signaling, cell differentiation, transcriptional regulation, cytokine
processing, and apoptosis (4, 19, 22). Calpain is also implicated in a
variety of pathological states, including ischemia, hypoxia,
vasospasm, Alzheimer's disease, sepsis, inflammation, and muscular
dystrophy (4, 19, 24, 29).
Exposure of endothelial cells to hypoxia alters many aspects of
endothelial cell function and metabolism, including membrane function,
cellular enzyme activity, cytokine secretion, protein synthesis, and
mRNA expression (1, 11, 14, 20, 28, 30). The effect of hypoxia on the
activity of the endothelial isoform of nitric oxide synthase (eNOS) is
controversial. McQuillan et al. (14) and Liao et al. (11) reported that
incubation of cultured endothelial cells in 0-3% O2
for 24-48 h resulted in the reduction of eNOS gene expression,
eNOS protein content, and eNOS activity. Xue et al. (28) demonstrated
that exposure of rats to 10% O2 for 2-4 wk induced
upregulation of eNOS expression in the pulmonary endothelium. However,
in this study, NOS activity in the particulate fraction of hypoxic lung
tissue homogenates (where the eNOS is located) remained unchanged.
Finally, Arnet et al. (1) reported that exposure to hypoxia (1%
O2) for 6-24 h increased eNOS mRNA and protein content
in cultured bovine aortic endothelial cells. However, the relative NOS
specific activity, i.e., NOS activity per NOS protein, decreased in
hypoxic endothelial cells.
The experiments of Xue et al. (28) and Arnet et al. (1) hint that
hypoxia modifies eNOS activity by a posttranslational mechanism.
Recently, we reported that hypoxia increases calpain activity and
expression in cultured porcine pulmonary artery endothelial cells
(PAEC) (24, 29) and that the increased calpain activity has a critical
role in the mobilization of L-arginine from proteins in
hypoxic cells (24). In the present study, we found that exposure of
PAEC to hypoxia for 24 h reduces eNOS specific activity but not eNOS
protein content, and we tested the hypothesis that calpain activation
is responsible for the hypoxia-induced decrease of eNOS activity
observed in porcine PAEC. Our results indicate that a calpain-mediated
mechanism is responsible for the hypoxia-induced decrease in eNOS
activity in PAEC and that this mechanism involves a reduction in heat
shock protein 90 (HSP90) content.
 |
METHODS |
Cell culture. Endothelial cells were obtained from the main
pulmonary artery of 6- to 7-mo-old pigs and were cultured as previously reported (24, 29). Third- to sixth-passage cells in monolayer culture
were maintained in RPMI 1640 medium containing 4% fetal bovine serum
and antibiotics (10 U/ml penicillin, 100 µg/ml streptomycin, 20 µg/ml gentamicin, and 2 µg/ml Fungizone) and were used 2 or 3 days
after confluence.
All monolayers were initially identified as endothelial cells by
phase-contrast microscopy. Selected dishes of cells were further
characterized by electron microscopy or by indirect immunofluorescent staining for factor VIII antigen or both. By use of these techniques, monolayer cultures were estimated to be pure endothelial cells.
Exposure to hypoxia. The confluent monolayers of PAEC were
exposed at 37°C to 0% O2-5% CO2-95%
N2 (hypoxia) or air-5% CO2 (normoxia) at 1 atmosphere absolute for 4-24 h as previously reported (24, 29) in
the presence and absence of the following agents: calpain inhibitor I
(10 µM); calpeptin (50 µM); tyrosine kinase inhibitor genistein (10 µM); tyrosine phosphatase inhibitor vanadate (20 µM); protein
kinase C inhibitors staurosporine (5 nM), calphostin C (200 nM), and
chelerythrine (1 µM); or geldanamycin (1 µg/ml), a specific
inhibitor of HSP90. The percentage of oxygen in the gas
leaving the exposure chambers was found to be <1.0% when cells were
exposed to 0% O2 and was 19-21% during exposure to
normoxic conditions. Oxygen tensions in the culture medium paralleled
those in the gas phase and were 7-12 and 140-155 mmHg,
respectively, in hypoxic and normoxic PAEC. Exposure to 0%
O2 for 24 h did not injure the cell monolayer as assessed
by phase-contrast microscopy and by quantitation of cell number and
protein content.
Determination of NOS activity. After exposure to normoxic or
hypoxic environments, the PAEC monolayers were scraped and homogenized in buffer A (50 mM Tris · HCl, pH 7.4, containing 0.1 mM each EDTA and EGTA, 1 mM phenylmethylsulfonyl
fluoride, 1.0 µg/ml leupeptin, and 10 µM calpain inhibitor I). The
homogenates were centrifuged at 100,000 g for 60 min at
4°C, and the total membrane pellet was resuspended in buffer
B (buffer A + 2.5 mM CaCl2). The resulting suspension was used for determination of eNOS activity by monitoring the formation of L-[3H]citrulline
from L-[3H]arginine (25). The total
membrane fractions (100 µg of protein) were incubated (total volume
0.4 ml) in buffer B containing 1 mM NADPH, 100 nM calmodulin,
10 µM tetrahydrobiopterin, and 5 µM combined L-arginine
and purified L-[3H]arginine (0.6 µCi; specific activity 69 Ci/mmol; NEN; Boston, MA) for 30 min at 37°C. Purification of
L-[3H]arginine and measurement of
L-[3H]citrulline formation were
carried out as described previously (25). The specific activity of NOS
is expressed as pmol
L-citrulline · min
1 · mg
protein
1 and reflects eNOS activity
because our cells do not exhibit basal or hypoxia-induced NOS activity.
Total membrane protein content was determined by the method of Lowry et
al. (13).
Western blot analysis of NOS protein and HSP90. After exposure
to normoxic or hypoxic environments, the PAEC were washed with phosphate-buffered solution and then lysed in boiling sample buffer (0.06 M Tris · HCl, 2% SDS, and 5% glycerol, pH
6.8). The lysates were boiled in a water bath for 5 min to remove
insoluble materials. The lysate proteins (20 µg) were fractionated on
7.5% SDS-PAGE gels and blotted onto polyvinylidene difluoride
membranes (Bio-Rad; Hercules, CA). The blots were incubated in blocking
solution (10 mM Tris · HCl, 0.2% nonfat milk, 100 mM
NaCl, and 0.1% Tween 20, pH 7.5) and then hybridized with 1:5,000
diluted monoclonal antibodies against eNOS or HSP90 (Transduction
Laboratories; Lexington, KY) at room temperature for 1 h. The bands
were detected using an immunochemiluminescence method, and the density
of the bands was quantitated using a Fluor-S-MultiImager (Bio-Rad).
Effect of calpain on eNOS activity and eNOS protein content. To
study possible posttranslational modification of eNOS activity by
calpain, PAEC total membrane fractions isolated in the absence of
protease inhibitors were incubated with calpain in vitro. The 200-µl
reaction mixture (300-400 µg of protein) contained 1 mM CaCl2, 5 mM cysteine, and 0.05-1.0 U/200 µl calpain
I (Calbiochem; San Diego, CA). After incubation at 30°C for 30 min,
the reactions were terminated by adding calpain inhibitor I to a final
concentration of 100 µM. A 10-µl aliquot was used for Western blot
analysis of eNOS protein content, and the rest of the reaction mixture was used for determination of eNOS activity by the method described previously.
Coprecipitation of HSP90 and caveolin-1 by anti-eNOS antibody.
PAEC exposed to normoxic or hypoxic conditions for 24 h were lysed in
ice-cold buffer containing 20 mM Tris · HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM Pefabloc,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µM calpain inhibitor
I, and 10 µg/ml pepstatin A. The cell lysates were centrifuged at
10,000 g for 20 min to remove insoluble material. Then 500 µl
of the lysates (500 µg protein) from normoxic or hypoxic cells were
incubated with 2.5 µg of anti-eNOS antibody at 4°C for 4-5
h. Protein A Sepharose (30 µl) was added, and the samples were
further incubated for 2 h at 4°C. The immunoprecipitates were
recovered by centrifugation and washed three times in buffer containing
50 mM Tris · HCl, pH 8.0, 150 mM NaCl, and 0.1%
Triton X-100. Immunoprecipitated proteins were eluted from the
Sepharose beads by boiling the samples for 5 min in 30 µl of SDS
immune-blotting sample buffer. The Sepharose beads were then pelleted
by centrifugation at 14,000 g, and eNOS, caveolin-1, and HSP90
protein contents in the supernatant were analyzed by Western blot as
described previously. Caveolin-1 was detected using a commercially
available monoclonal antibody (Transduction Laboratories).
Determination of NOS activity in anti-eNOS antibody
immunoprecipitates (immuno-NOS assay). The immuno-NOS assay was
done according to the method described by Garcia-Cardena et al. (8).
Briefly, eNOS antibody-induced immunoprecipitates were obtained from
PAEC exposed to normoxic and hypoxic conditions as described in
Coprecipitation of HSP90 and caveolin-1 by anti-eNOS antibody,
except that the detergent was 10 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate instead of 1% Triton X-100 and 0.1% SDS. The protein
A-Sepharose beads were resuspended in 0.6 ml of NOS assay buffer
(buffer B). eNOS activities in the presence or absence of HSP90
(30 µg/ml) or
NG-monomethyl-L-arginine (1 mM) were
determined as described in Determination of NOS
activity.
Data analysis. In each experiment, experimental and control
endothelial cells were matched for cell line, age, seeding density, number of passages, and number of days postconfluence to avoid variation in tissue culture factors that can influence the measurement of NOS activity and NOS protein content. Results are shown as means ± SE for n experiments. Student's t-test was used to
determine the significance of differences between means of hypoxic and
normoxic groups, and P < 0.05 was considered significant.
 |
RESULTS |
Exposure of PAEC to hypoxia for 4-24 h resulted in a
time-dependent decrease of eNOS activity (Fig.
1), whereas eNOS protein content was
unchanged in hypoxic PAEC (Fig. 2). To
determine whether the hypoxia-induced decrease of eNOS activity was
associated with calpain, PAEC were exposed to hypoxia for 24 h in the
presence and absence of calpain inhibitor I (10 µM). As indicated in
Fig. 3, calpain inhibitor I prevents the
loss of eNOS activity in PAEC exposed to hypoxia for 24 h. Similar
results were observed with calpeptin, another specific inhibitor of
calpain.

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Fig. 1.
Effect of hypoxia on endothelial nitric oxide synthase (eNOS) activity
in pulmonary artery endothelial cells (PAEC). Cells were exposed to
normoxia (control) or hypoxia for 4-24 h, after which eNOS
activity was determined as described in METHODS. Results
are expressed as means ± SE; n = 5 experiments.
* P < 0.01 vs. control.
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Fig. 2.
Effect of hypoxia on eNOS protein content in PAEC. Cells were incubated
in presence of normoxia (control) for 24 h or in presence of hypoxia
for 2-24 h, after which eNOS protein was measured by Western blot
analysis as described in METHODS. A: representative
Western blot of eNOS protein content. B: bar graph depicting
eNOS protein contents quantified by scanning densitometry. Comparable
results were obtained in 4 separate experiments.
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Fig. 3.
Effect of calpain inhibitor I on hypoxia-induced inhibition of eNOS
activity in PAEC. Cells were exposed to normoxia or hypoxia for 24 h in
presence and absence (control) of calpain inhibitor I (10 µM) after
which eNOS activity was assayed as described in METHODS.
Results are expressed as means ± SE; n = 4 experiments.
* P < 0.01 vs. normoxic control.
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To confirm that calpain can decrease eNOS activity in PAEC and to
ascertain whether this is a direct effect of calpain on eNOS, membrane
fractions of PAEC were incubated with calpain I (0.05-1.0 U/200
µl) in vitro for 30 min at 30°C. Incubation of PAEC membranes
with calpain I resulted in a dose-dependent decrease of eNOS activity
(Fig. 4). Reduction in eNOS activity
occurred with as little as 0.1 U calpain I/200 µl. In contrast, eNOS
protein content was not affected by calpain I concentrations of up to 0.5 U/200 µl. eNOS protein content was decreased in the presence of
1.0 U/200 µl of calpain I, a concentration that is 10-fold higher
than that which we have observed in hypoxic PAEC (10). Incubation with
calpain inhibitor I prevented the decreases in eNOS activity and
content (Fig. 4).

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Fig. 4.
Effect of calpain I on eNOS activity and eNOS protein content in total
membrane fractions prepared from PAEC. Membranes of PAEC were incubated
with and without (control) calpain I (0.05-1.0 U/200 µl) under
normoxic conditions in vitro for 30 min at 30°C, after which eNOS
activity and eNOS protein contents were measured as described in
METHODS. A: representative Western blot of eNOS
protein. B: bar graph depicting eNOS protein content quantified
by scanning densitometry. C: bar graph depicting eNOS activity.
Results are expressed as means ± SE; n = 3 experiments. In experiments involving calpain inhibitor I, the calpain
concentration was 1.0 U/200 µl, and concentration of inhibitor was
100 µM. * P < 0.05 and ** P < 0.01 vs. control.
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Caveolin and HSP90 have been reported to bind to eNOS and to regulate
its catalytic activity (7, 8, 16). To determine whether the
calpain-mediated inhibition of eNOS activity that we observed in
hypoxic PAEC is associated with a change in the content of one of these
regulatory proteins, we measured caveolin-1 and HSP90 protein contents
in normoxic and hypoxic PAEC. Exposure to hypoxia did not affect
caveolin-1 content in PAEC (data not shown). In contrast, exposure to
hypoxia for 2-24 h resulted in a time-dependent decrease of HSP90
protein content that parallels the time course of the decrease of eNOS
protein content in hypoxic cells (Fig. 5).
Calpain inhibitor I prevented the hypoxia-induced decrease of HSP90
protein content (Fig. 6). Similar results
were obtained with calpeptin.

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Fig. 5.
Effect of hypoxia on heat shock protein 90 (HSP90) content. PAEC were
exposed to hypoxia for 2-24 h after which HSP90 content was
measured as described in METHODS. A: representative
Western blot of HSP90. B: bar graph depicting HSP90 content.
Results are expressed as means ± SE; n = 5 experiments.
* P < 0.05 vs. control (0-h exposure).
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Fig. 6.
Effect of calpain inhibitor I on hypoxia-induced decrease of HSP90
content. PAEC were exposed to normoxia or hypoxia for 24 h in presence
and absence (control) of calpain inhibitor I (10 µM) after which
HSP90 content was measured as described in METHODS.
A: representative Western blot of HSP90. B: bar graph
depicting HSP90 content. Results are expressed as means ± SE;
n = 4 experiments. * P < 0.05 vs.
normoxic control.
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To study whether the protein-protein interactions between eNOS and the
two regulatory proteins, caveolin-1 and HSP90, were affected by
hypoxia, the lysates from PAEC exposed to normoxia or hypoxia were
immunoprecipitated with antibody directed against eNOS.
Immunoprecipitation of eNOS from PAEC lysates resulted in the
coprecipitation of caveolin-1 and HSP90. Exposure to hypoxia did not
alter the caveolin-1 content coprecipitated with eNOS (not shown), but
exposure to hypoxia did significantly reduce the amount of HSP90
coprecipitated with eNOS (Fig. 7). Purified HSP90 expressed in Escherichia coli added back to the
eNOS-HSP90 complex immunoprecipitated by anti-eNOS antibody reversed
the decrease of eNOS activity in the immunoprecipitates from hypoxic PAEC (Fig. 8).

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Fig. 7.
Effect of hypoxia on content of HSP90 associated with eNOS. Lysates of
PAEC exposed to normoxic (control) and hypoxic conditions for 24 h were
immunoprecipitated (IP) by nonimmune IgG or anti-eNOS antibody, and
eNOS and HSP90 contents in precipitates were measured as described in
METHODS. A: representative Western blot of
immunoprecipitates. B: bar graph depicting ratio of HSP90 to
eNOS. In these experiments, eNOS protein contents precipitated were the
same in control and hypoxic groups. Results are expressed as means ± SE; n = 3 experiments. * P < 0.01 vs. control.
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Fig. 8.
Effect of HSP90 on hypoxia-induced inhibition of eNOS activity in
immunoprecipated pellets. Lysates of PAEC exposed to normoxic and
hypoxic conditions for 24 h were immunoprecipitated by
anti-eNOS antibody, and eNOS activities in pellets were measured in
presence of purified HSP90 (30 µg/ml) or of
NG-monomethyl-L-arginine
(L-NMMA, 1 mM) as described in METHODS.
Controls were assayed in absence of HSP90 and
L-NMMA. Results are expressed as means ± SE of
L-citrulline generated by reaction mixture; n = 4 experiments. * P < 0.01 vs. normoxic control.
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To evaluate further the relationship between HSP90 and eNOS activity,
we used geldanamycin, a specific inhibitor of HSP90. Geldanamycin binds
to the nucleotide-binding site of HSP90 and specifically blocks HSP90
function (9). Incubation of PAEC with geldanamycin not only resulted in
a dose-dependent decrease of eNOS activity, but it also prevented the
hypoxia-induced decrease of eNOS activity (Fig.
9). Geldanamycin did not inhibit eNOS
activity when added directly to the total membrane fraction of PAEC.

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Fig. 9.
Effect of geldanamycin on eNOS activity and hypoxia-induced inhibition
of eNOS activity. A: PAEC under normoxic conditions were
treated with varying concentrations of geldanamycin for 24 h after
which eNOS activity was measured. B: PAEC were exposed to
normoxia or hypoxia for 24 h in presence and absence (control) of
geldanamycin (1 µg/ml) after which eNOS activities were measured.
Results are expressed as means ± SE; n = 4 experiments.
*P < 0.01 vs. normoxic control.
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Several groups have reported that tyrosine phosphorylation and serine
phosphorylation of eNOS alter its activity (2, 8). To investigate
whether tyrosine phosphorylation or serine phosphorylation is
responsible for the hypoxia-induced loss of eNOS activity observed in
the present study, PAEC were exposed to normoxia or hypoxia for 24 h in
the presence and absence of the tyrosine kinase inhibitor genistein,
the tyrosine phosphatase inhibitor vanadate, and the protein kinase C
inhibitors staurosporine, calphostin C, and chelerythrine. In contrast
to geldanamycin and inhibitors of calpain, i.e., calpain inhibitor I
and calpeptin, none of these inhibitors prevented the hypoxia-induced
loss of eNOS activity in PAEC (Table 1).
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Table 1.
Effects of staurosporine, calphostin C, chelerythrine, genistein, and
vanadate on hypoxia-induced reduction of eNOS activity in PAEC
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 |
DISCUSSION |
In the present study, we have shown that hypoxia causes a
time-dependent decrease in eNOS activity in cultured PAEC that is unaccompanied by changes in eNOS protein content. These results are
consistent with the thesis that hypoxia inhibits eNOS activity by a
posttranslational mechanism. Xue et al. (28) and Arnet et al. (1) also
suggested that hypoxia modified eNOS activity by a posttranslational
mechanism. There are several possible mechanisms by which eNOS function
might be regulated posttranslationally. First, eNOS protein can be
phosphorylated by protein kinase C, and serine phosphorylation has been
shown to downregulate eNOS activity (2). This seems unlikely to account
for our results because three specific inhibitors of protein kinase C,
staurosporine, calphostin C, and chelerythrine, did not have any effect
on the hypoxia-induced loss of eNOS activity in our cells. Second,
tyrosine phosphorylation of eNOS may affect its activity (8, 26). However, neither genistein nor vanadate affected the hypoxia-induced decrease of eNOS activity. Moreover, hypoxia did not alter tyrosine kinase and tyrosine phosphatase activities in PAEC (unpublished data).
Third, eNOS can be myristoylated or palmitoylated (3, 12). However,
neither of these modifications has been reported to change the in vitro
catalytic activity of eNOS (3, 12).
Our results indicate that calpain is responsible for the
hypoxia-mediated decrease in eNOS activity in porcine PAEC. The
specific calpain inhibitors calpain inhibitor I and calpeptin prevented the hypoxia-induced loss of eNOS activity. Moreover, incubation of PAEC
total membrane fractions with concentrations of calpain I observed in
hypoxic PAEC resulted in decreases in eNOS activity in the absence of
changes in eNOS protein content that were prevented by calpain
inhibitor I.
To explore further the mechanism by which calpain mediates the decrease
in eNOS activity observed in hypoxic PAEC, we considered the
possibility that calpain activation was either resulting in the
inhibition of an activator of eNOS or causing the activation of an
inhibitor of eNOS. There are several reports that eNOS activity can be
regulated by the interaction between the eNOS protein and other
proteins such as caveolin-1 (8, 16), calmodulin (16), and HSP90 (7).
Interaction of eNOS with caveolin leads to inhibition of eNOS activity.
The binding of Ca2+/calmodulin to eNOS disrupts this
inhibitory eNOS-caveolin complex, leading to enzyme activation.
However, in the present study, hypoxia did not change the caveolin-1
content that was coimmunoprecipitated with eNOS protein. An alteration
of calmodulin content is unlikely to be responsible for the decreased
eNOS activity in hypoxic PAEC because 100 nM calmodulin was included in
the reaction mixture used to measure eNOS activity in our in vitro
assay. Recently, Garcia-Cardena et al. (7) found that binding of HSP90
to eNOS enhances the activation of eNOS. Our results indicate that
hypoxia caused a time-dependent decrease of HSP90 that corresponded to the time course of change of eNOS activity in the hypoxic PAEC. Calpain
inhibitor I and calpeptin prevented this hypoxia-induced decrease of
HSP90 protein content. Moreover, the HSP90 content that was
coprecipitated by the anti-eNOS antibody was reduced in hypoxic PAEC
compared with normoxic cells. Repletion of HSP90 in the anti-eNOS
antibody-induced-immunoprecipitates restored eNOS activity in the
pellets from hypoxic PAEC. Finally, the specific inhibitor of HSP90
(geldanamycin) mimicked the effect of hypoxia, i.e., it decreased eNOS
activity to the same degree as exposure to hypoxia for 24 h and
prevented further decrease of eNOS activity by subsequent exposure to
hypoxia. Taken together, these results indicate that a reduction in
HSP90 is responsible for the calpain-mediated decrease of eNOS activity
observed in hypoxic PAEC.
Pulmonary endothelial cells are an important source of NO, and NO is an
important endogenous vasodilator that contributes to the low vascular
resistance in the pulmonary circulation (23). Several reports indicate
that hypoxia causes a decrease in the synthesis and/or release of NO
from pulmonary endothelial cells (18, 27). Moreover, mice with targeted
disruption of the eNOS gene become hyperresponsive to mild hypoxia (5).
Our study suggests calpain is involved in hypoxia-induced reduction of
eNOS activity. Ruetten and Thiermermann (19) have reported that calpain has an important role in the release of NO in the condition of sepsis.
Thus calpain, serving as a signal transduction molecule, may have an
important role in the regulation of vascular function, especially under
hypoxic conditions.
 |
ACKNOWLEDGEMENTS |
We thank Humberto Herrera for assistance with tissue culture,
Adelaide Heimer for secretarial support, and Janet Wootten for editorial 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-52136.
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: E. R. Block,
Research Service (151), Malcom Randall Dept. of Veterans Affairs
Medical Center, 1601 S.W. Archer Road, Gainesville, FL
32608-1197.
Received 29 November 1999; accepted in final form 11 January 2000.
 |
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