From the Division of Pediatric Surgery, the
Departments of § Pharmacology & Toxicology,
Physiology, and ¶¶ Biochemistry,
the ¶ Cardiovascular Research Center, and the ** Medical Scientist
Training Program, Medical College of Wisconsin, Milwaukee, Wisconsin
53226 and the §§ Department of Pharmacology,
Boyer Center for Molecular Medicine, Yale University School of
Medicine, New Haven, Connecticut 06536
Received for publication, February 14, 2001, and in revised form, March 13, 2001
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ABSTRACT |
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The balance of nitric oxide (·NO)
and superoxide anion (O Nitric oxide (·NO) and superoxide anion (O Emerging evidence suggests that under pathological conditions eNOS may
also generate O L-Nitroargininemethylester
(L-NAME),
NG-monomethyl-L-arginine·monoacetate,
(L-NMA), dimethyl sulfoxide (Me2SO),
geldanamycin (GA), antibiotics/mycotics, trypsin-EDTA, Hank's balanced
salts solution (HBSS), L-arginine, KI, glacial acetic acid,
sodium nitrite, ferricytochrome c, superoxide dismutase (SOD), NaF,
sodium deoxycholate, SDS, 4-(2-aminoethyl benzene) sulfonyl fluoride
hydrochoride, sodium orthovanadate, leupeptin, pepstatin A, aprotinin,
protein A-Sepharose were from Sigma Chemical Company (St. Louis, MO). L-Sepiapterin was from Alexis Biochemicals (San Diego, CA).
(HCl, NaOH, were from Fisher Scientific (Pittsburg, PA). RPMI 1640 was from Life Technologies, Inc. (Grand Island, NY). Tris-HCl and NaCl were
from Baker (Phillipsburg, NJ). Hydroethidine was from Polysciences,
Inc. (Warrington, PA). Triton X-100 was from Lab Chem (Pittsburg, PA).
A23187 was from CalBiochem (San Diego, CA). Laemmli buffer,
polyacrylamide, nitrocellulose membranes were from Bio-Rad (Hercules,
CA). Fetal bovine serum was from HyClone (Logan, UT). ECL reagents were
from Amersham Pharmacia Biotech. X-OMAT film was from Kodak (Rochester,
NY). H32 antibody was from BioMol (Plymouth Meeting, PA) and Jennifer
Pollock (Medical College of Georgia, Augusta, GA). Anti-phospho-eNOS
(Ser-1177) was from Cell Signaling Technology (Beverly, MA).
Endothelial Cell Culture--
Bovine coronary endothelial cells
(BCEC) were provided by William B. Campbell (Milwaukee, WI). BCEC were
cultured in RPMI 1640 media containing 20% fetal bovine serum,
antibiotics, mycotics, rhFGF (10 ng/ml), and heparin (5 units/ml). BCEC
were passaged with trypsin-EDTA and used for experiments between
passage 5-7.
Experimental Protocol--
The protocol for the experiments was
as follows. For nitrite and O Measurements of Nitrite--
After the third wash, V-treated,
L-NMA-treated, GA-treated, and GA-L-NMA-treated
test cultures in 6-well plates were incubated with 0.75 ml HBSS
containing A23187 (5 µM) and L-arginine (25 µM) for 30 min. Nitrite was quantified using Sievers NOA
analyzer as described (10). Each experiment was performed in
triplicate; nitrites were analyzed in duplicate or triplicate, and cell
protein for each well was determined in duplicate.
Measurements of Superoxide Anion--
After the final HBSS wash,
the test groups in 6-well dishes (V, L-NAME, GA, and
GA+L-NAME) were incubated with 1 ml of HBSS containing
ferricytochrome c (50 µM) and A23187 (5 µM) with and without L-NAME (1 mM) for 30 min. Superoxide anion production was calculated
from the absorbance of ferricytochrome c at 550 nm. The
release of O Detection of Endothelial Superoxide Anion Generation in Isolated
Carotid Arteries--
Canine carotid arteries were obtained from adult
mongrel dogs. The vessel was removed, transferred to a physiologic
saline solution and adventitia was cleared. The artery was sectioned into segments at least 2 cm long and placed in RPMI 1640 media. After
two washings in RMPI 1640 (to remove adherent blood cells), vessels
were placed in organ culture. Artery segments were incubated with V or
GA as above, washed free of vehicle and GA, and then incubated with
hydroethidine (10 µM) for 30 min. Hydroethidine is taken
up by cells and in the presence of O Western Analysis, Immunoprecipitation, and
Immunoblotting--
Phospho-eNOS(Ser-1177) and eNOS levels were
determined using the manufacturer's protocol. Briefly, confluent BCEC
cultures in 60-mm dishes were pretreated with GA (10 µg/ml) for 30 min. The cultures were washed three times with HBSS and then stimulated with A23187 (5 µM) in HBSS for 10 min at 37 °C with
gentle horizontal rotation (50 rpm). After incubation, the HBSS
solutions were removed by aspiration, and cell proteins were harvested
in 200 µl of SDS sample buffer. Aliquots (50 µl) were heated
(95 °C, 5 min) and stored on ice until loaded (20 µl/lane) on 7%
SDS-PAGE. The proteins were transferred to nitrocellulose membranes and blotted with anti-phospho-eNOS(Ser-1177) and anti-eNOS (9D10, Zymed Laboratories Inc.) overnight at 4 °C. Bands
were visualized using the appropriate horseradish peroxidase
(HRP)-linked secondary antibodies and ECL reagents.
Co-precipitation studies for determining the interaction of Hsp90 with
eNOS were performed on confluent BCEC cultures in 100-mm dishes. The
four test groups were V, GA, V+A23187, and GA+A23187. After
preincubation with GA, the test groups were washed three times with 5 ml of HBSS and then incubated at 37 °C in a tissue culture incubator
in 5 ml of HBSS containing L-arginine (10 µM) with and without A23187 (5 µM). After 10 min, the HBSS
solutions were removed, and the cells were lysed in modified RIPA
buffer as described (2). The samples were sonicated two times at a power setting of 1.25-1.5 for 30 s on a Fisher Scientific
Dismembrater (Model 550) fitted with an Ultrasonic Convertor (Model No.
CL4 with a frequency of 20 kHz) probe. For this procedure it is
important to adjust the power settings of the unit to 5-10% of
maximum power output because higher power outputs disrupt eNOS
interactions with other cell proteins. Cell debris was removed from the
cell lysates, and eNOS was immunoprecipitated as described (2). The
immunoprecipitated proteins were separated by SDS-PAGE (7.5-15%) and
transblotted onto nitrocellulose. The membrane was blocked with 5%
nonfat milk in TBS-Tween (0.1%) and then immunoblotted for eNOS and
Hsp90 using 9D10 (33-4600, 1:1000) from Zymed Laboratories Inc., and H38220 (1:1000) from Transduction Laboratories,
respectively. Bands were visualized with the appropriate
anti-immunoglobulin-HRP conjugate from Sigma using ECL reagents and
Kodak X-OMAT film. The autoradiograms were imaged with Adobe PhotoShop
v5.5 and UMAX Magicscan v4.4 software, and the relative band densities
were quantified using NIH Image 1.62.
Statistical Analysis--
Nitrite and superoxide anion data were
examined by one-way analysis of variance to determine whether the
variances and means were significantly different. The Newman-Kuel
post-hoc test was employed to determine the level of significance
between means (**, p < 0.001 and *, p < 0.01).
Geldanamycin (GA) significantly inhibited A23187-stimulated
nitrite production by BCEC cultures incubated in HBSS containing low
concentrations of L-arginine (25 µM) (Fig.
1A). In contrast, A23187
stimulation of GA-treated BCEC cultures significantly increased the
release of O
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
= 21,000 M
1
cm
1). Data were compared with O
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
Effects of GA on A23187-stimulated
eNOS-dependent nitrate production and superoxide anion generation.
A, GA inhibits eNOS-dependent A23187-stimulated
nitrite production. The bar chart shows that GA (10 µg/ml,
30 min) significantly inhibits A23187-stimulated (5 µM,
30 min) eNOS-dependent nitrite production by bovine
coronary endothelial cell cultures (*, p < 0.001;
n = 4). Data are shown as mean ± S.E. and pmol/mg
cell protein. eNOS-dependent nitrite production was
determined by calculating the difference in nitrite production between
A23187-stimulated control and GA cultures and
L-NMA-inhibited (1 mM, 30 min) control and GA
cultures in HBSS containing L-arginine (25 µM). B, GA increases stimulated superoxide
anion generation. The bar chart shows that GA significantly
increases the release of O
The above observations demonstrate that GA increases the release of
O
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Serine phosphorylation of eNOS at Ser-1177 (human) and Ser-1179
(bovine) has been shown to correlate directly with increased ·NO
production (13). Treatment of BCEC cultures with A23187 increased
phospho-eNOS(Ser-1179) levels compared with the levels seen in
V-treated cultures (Fig. 3A,
top panel). Pretreatment with GA alone increased
phospho-eNOS levels (>2.5-fold) compared with V-treated cultures.
A23187 stimulation of GA-treated cultures also increased phospho-eNOS.
These data indicate that GA increases serine phosphorylation of eNOS, a
marker of increased eNOS activity (2, 14). The observation that A23187
simultaneously increases L-NAME-inhibitable O
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Hsp90 serves many functions within cells (see Ref. 15 for
review). It assists in protein folding by first binding and then changing the conformation of the Hsp90 bound to the client proteins. In
this respect, GA has been a useful tool for distinguishing between the
steps of association and conformation in protecting enzyme function
(16). To begin to understand how each step plays a role in preserving
eNOS function co-precipitation studies of eNOS were performed on
V-treated, V-treated + A23187, GA-treated, and GA-treated + A23187-
stimulated test groups. None of these treatments affected eNOS levels
(Fig. 3B, upper panel). Independently GA and
A23187 slightly increased the levels of Hsp90 that co-precipitated with eNOS (Fig. 3B, lower panel, lanes
2 and 3, respectively). In combination however, they
markedly increased the amount of Hsp90 that could be
co-immunoprecipitated with eNOS (Fig. 3B, lower
panel, lane 4). These observations are consistent
with the report showing GA increases Hsp90 binding to heat-denatured
luciferase but not necessarily enzyme activity (16). On the basis that GA inhibits conformational changes in Hsp90 and changes eNOS product formation from ·NO to O
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DISCUSSION |
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This study shows that altering the interaction of Hsp90 with eNOS
uncouples enzyme activity resulting in increased O
By measuring the effects of GA on nitrite and O
Superoxide anion can be generated by eNOS at either the NADPH reductase
domain or the arginine oxidase domain (8, 17-20). Xenobiotics such as
paraquat, lucigenin, and adriamycin as well as excess FAD and FMN
increase O
On the basis that GA is a specific inhibitor of Hsp90 activity and BH4
supplementation did not inhibit the effects of GA on eNOS-dependent O
In conclusion, Hsp90 modulates eNOS product formation. When Hsp90 is
bound to eNOS and can change conformation, eNOS generates ·NO upon
stimulation. When Hsp90 is bound to eNOS and conformational changes are
restricted or impaired, eNOS generates O
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ACKNOWLEDGEMENTS |
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We thank Drs. Keith T. Oldham and Karen Guice for their helpful suggestions and support; the Medical Scientist Training Program of MCW for funding Eric R. Gross' summer rotation in Dr. Pritchard's laboratory; Jennifer Pollock for providing anti-eNOS antibodies; and Neal Hogg, Ph.D. and the Free Radical Research Center for assistance in nitrite measurements by ozone chemiluminescence.
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FOOTNOTES |
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* This work was supported in part by Grants HL61414 (to K. A. P.), HL54075 (to J. E. B.), AHA 0020453Z (to Y. S.), and HL 57665, HL 61371, and HL 64793 (to W. C. S.) from the National Institutes of Health.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.
To whom correspondence should be addressed: Medical College of
Wisconsin, Division of Pediatric Surgery, Pharmacology & Toxicology, Cardiovascular Research Center, 8701 Watertown Plank Rd., Milwaukee, WI
53226. Tel.: 414-456-5615; Fax: 414-456-6473; E-mail:
KPRITCH@mcw.edu.
An Established Investigator of the American Heart Association.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.C100084200
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ABBREVIATIONS |
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The abbreviations used are: eNOS, endothelial nitric-oxide synthase; GA, geldanamycin; BCEC, bovine coronary endothelial cells; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; L-NAME, L-nitroargininemethylester, L-NMA, NG-monomethyl-L-arginine·monoacetate; SOD, superoxide dismutase; V, vehicle; RIPA, radioimmune precipitation buffer.
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