© 2000 American Heart Association, Inc.
Vascular Biology |
Stimulation of Receptor-Mediated Nitric Oxide Production by Vanadate
From the Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa.
Correspondence to Larry P. Solomonson, Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, 12901 Bruce B. Downs Blvd, MDC Box 7, Tampa, FL 33612-4799. E-mail lsolomon{at}hsc.usf.edu
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Abstract |
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Abstract—Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore– (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to
125 kDa and 2 minor bands corresponding to 105
and 75 kDa after treatment with vanadate/Bk. No tyrosine
phosphorylation of eNOS after treatment with
vanadate/Bk was observed. Geldanamycin, an inhibitor of
heat shock protein 90, also inhibited the enhancement of NO
production by vanadate/Bk or vanadate/A23187, and there was an
increase in the amount of heat shock protein 90 that
coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These
results show that there is a clear link between tyrosine
phosphorylation and stimulation of eNO
production, which does not appear to involve direct
modification of eNOS, changes in eNOS levels, or compartmentation, but
rather appears to be due to changes in proteins associating with eNOS,
thereby enhancing the state of activation of eNOS.
Key Words: nitric oxide • endothelial • vanadate • bradykinin • tyrosine phosphorylation
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Introduction |
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Endothelial nitric oxide synthase (eNOS) plays a key role in vasoregulation through the highly regulated production of eNO, which diffuses to the smooth muscle layer, causing relaxation. Several studies have suggested a linkage between phosphorylation/dephosphorylation events and eNO production. An early study by Michel et al1 demonstrated that short-term incubation of endothelial cells with bradykinin (Bk) or calcium ionophore A23187 led to serine phosphorylation of eNOS, which appeared to be associated with intracellular translocation of eNOS.2 More recent studies have demonstrated that Akt (protein kinase B) activates eNOS by phosphorylation of a specific serine residue.3 4 5 The involvement of tyrosine phosphorylation in eNOS regulation has also been studied. Exposure of endothelial cells to vanadate resulted in a low level of tyrosine phosphorylation, compared with a much greater level of serine phosphorylation.6 The in vitro activity measured on immunoprecipitates of eNOS suggested that tyrosine phosphorylation reduced the activity of eNOS by about one half.6 Fleming et al7 found a low level of basal tyrosine phosphorylation of eNOS, which was lost after treatment of endothelial cells with a phosphotyrosine phosphatase inhibitor (phenylarsine oxide). Venema et al8 observed neither basal nor Bk-mediated stimulation of tyrosine phosphorylation of eNOS but did find that Bk activation of eNOS was accompanied by tyrosine phosphorylation of an eNOS-associated protein (ENAP-1). Activation of eNOS by flow-mediated shear stress appears to involve tyrosine phosphorylation, the activation being reduced by tyrosine kinase inhibitors and stimulated by inhibitors of phosphotyrosine phosphatases.7 9 10 11 Corson et al9 proposed that shear stress–mediated eNOS activity might be regulated by phosphorylation of the enzyme. They found an increase in serine/threonine phosphorylation, but not tyrosine phosphorylation, of eNOS after application of shear stress. Thus, the precise role of tyrosine phosphorylation in the regulation of eNOS activity is still uncertain.
In the present study, we examined the effect of phosphotyrosine phosphatase inhibition by sodium orthovanadate (vanadate) on eNO production and found an unexpectedly large and sustained increase in the amount of eNO produced after Bk- or calcium ionophore–mediated activation of eNOS. Although eNOS does not appear to be directly tyrosine-phosphorylated under these conditions, changes in the level of certain eNOS-associated phosphotyrosine-containing and other proteins suggest a role for tyrosine phosphorylation/dephosphorylation in the physiological regulation of eNOS activity.
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Methods |
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Cell Culture
Endothelial cells used in this study were isolated from bovine aortas (BAECs) according to a standard method12 and verified as endothelial by positive immunostaining for von Willebrand factor, negative staining for muscle actin, and morphological examination by electron microscopy.13 Cells were routinely seeded in 12-well culture plates (Corning/Costar) at dilutions of 1:3 to 1:5 and grown to confluence in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U penicillin, 100 µg streptomycin, 0.25 µg amphotericin B, and 50 µg gentamycin per milliliter at 37°C in an atmosphere of 95% air/5% CO2.
Treatment Conditions
Cells were used 2 to 3 days after reaching confluence at a
density of 1.5 to 1.7x105
cells/cm2 and >98% viability by the trypan blue
dye-exclusion test. Cell monolayers in 12-well cluster dishes were
rinsed 3 times with standard PBS at 37°C, followed by the addition of
0.7 mL of Dulbecco’s modified Eagle’s medium containing antibiotics
but no serum to each well. The NOS inhibitor
NG-nitro-L-arginine
methyl ester (L-NAME) was added, where indicated,
to a final concentration of 1 mmol/L and incubated for 30 minutes
at 37°C before addition of the test compounds. Phosphatase or kinase
inhibitors were added 5 minutes before eNOS activation with
Bk, A23187, vanadate, vanadate/Bk, or vanadate/A23187. Incubations were
carried out at 37°C and test compounds were present throughout
the incubation period. Solutions of sodium orthovanadate at a
concentration of 5 mmol/L were prepared by heating at 95°C for
10 minutes, and actual concentrations of orthovanadate were determined
by spectrophotometric measurement at 260 nm by using a millimolar
extinction coefficient of 3.55.14
Assay for NO as Nitrite in Culture Medium
The method for measuring nitrite, a stable reaction product
of NO, involves the conversion of 2,3-diaminonaphthalene to the highly
fluorescent 1(H)-naphthotriazole through specific
reaction with nitrite under acidic conditions.15 Cell
counts were performed on a sampling of wells by using a microscope with
an eyepiece micrometer, and the average was used in
converting the readings to a per-106-cell basis.
Fluorescence intensity was determined at room temperature with
a Jasco FP-770 spectrofluorometer.
Preparation of Caveolin-Enriched Membrane Fractions
The method of Song et al16 was used to prepare
caveolae from BAECs. All steps were carried out at 4°C. In brief,
confluent cell monolayers were treated with various agents, washed with
PBS, and suspended in 500 mmol/L
Na2CO3, pH 11. The alkaline
cell suspension was lysed with a Dounce homogenizer
followed by further homogenization with a Polytron
tissue homogenizer and brief sonication, as described
by Song et al.16 One milliliter of each lysate was mixed
with an equal volume of 90% sucrose, transferred to the bottom of a
centrifuge tube, and overlaid with a step gradient consisting
of 1.0 mL of 35% sucrose, 0.5 mL of 25% sucrose, 0.5 mL of 15%
sucrose, and 0.5 mL of 5% sucrose, all with the same buffer
composition as the 45% sucrose sample solution. Tubes were
centrifuged in an SW60 Ti rotor at 40 000 rpm for 17 hours.
Fractions of 0.4 mL were collected from the top of the gradient. The
caveolar peak corresponded to fraction 3.
Preparation and Processing of Cell Lysates
After the indicated incubation period, cells were rinsed 3 times
with cold PBS and stored at -80°C until processed. Lysates were
prepared by scraping cells from each well into 250 µL of
cell-disruption buffer containing 20 mmol/L HEPES (pH 7.5); 0.1
mol/L NaCl; 5 µg/mL each of pepstatin A, leupeptin, and trypsin
inhibitor; 0.5 mmol/L PMSF; 0.1 mmol/L DTT;
1 mmol/L sodium orthovanadate; and 10 mmol/L KF. Three rounds
of freeze/thaw or three 6-second bursts of sonication with a microprobe
at a 25-W output were sufficient to lyse >90% of the cells, as judged
microscopically. Cell lysates were used directly, separated into
caveolar fractions (see above), or separated into membrane and
cytosolic fractions by 15 minutes of centrifugation at
100 000g in a TL-100 ultracentrifuge with a
fixed-angle rotor. Protein determinations were done using the
bicinchoninic acid protein assay kit (Pierce) with bovine serum
albumin as the standard.
Immunodetection of Proteins and Immunoprecipitation
For determination of eNOS protein levels, aliquots of cell
lysates or fractions were either directly adsorbed to nitrocellulose
membranes with a dot-blot apparatus (Bio-Rad) or separated
by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and
electroblotted onto nitrocellulose membranes. For immunoprecipitations,
cell lysates (100 µg protein) were incubated with a specific
antibody (1 µg) for 1 hour at 4°C, followed by 1 hour at 4°C
with protein A/G Sepharose (Sigma). Precipitated proteins were
solubilized with SDS-PAGE sample buffer and electrophoretically
separated. Blots were blocked in 1% gelatin or 5% nonfat dry milk in
10 mmol/L Tris · HCl (pH 7.5), 0.1 mol/L NaCl, and 0.2%
Tween 20 and then incubated, where indicated, with 1:1000 dilutions of
monoclonal antibodies specific for eNOS (Transduction Labs), caveolin-1
(Transduction Labs), protein tyrosine phosphate (clone PY20, Zymed
Labs), or heat shock protein 90 (hsp90; StressGen Labs). Horseradish
peroxidase–conjugated IgG (Transduction Labs) was used to detect the
respective primary monoclonal antibody by means of a horseradish
peroxidase–activated chemiluminescent substrate (enhanced
chemiluminescence, Amersham). Densitometric measurements on scanned
images were completed by using ImageQuaNT software (Molecular
Dynamics).
In Vitro Assay for eNOS Activity
The possible direct effect of sodium orthovanadate on eNOS
activity in lysates of endothelial cells was tested by
using an eNOS assay based on the conversion of
[3H]L-arginine to
[3H]citrulline.17 Assay mixtures
contained 25 mmol/L HEPES (pH 7.4), 3 µmol/L
tetrahydrobiopterin, 1 µmol/L FAD, 1 µmol/L flavin
mononucleotide, 1 mmol/L NADPH, 0.6 mmol/L
CaCl2, 0.1 µmol/L calmodulin,
and 20 µCi/mL [3H]L-arginine (60
Ci/mmol). Assays were run for 30 and 60 minutes in the presence or
absence of 1 mmol/L sodium orthovanadate at 37°C, and activity
was compared with background levels in the absence of calcium or in the
presence of the NOS inhibitor L-NAME. Lysates
from cells treated with Bk and/or vanadate were also assayed for eNOS
activity to determine whether the treatments resulted in any change in
specific activity of eNOS.
Statistical Analysis
Data are expressed as mean±SEM. At least triplicate
determinations were performed for quantitative analyses.
Student’s t test was used for evaluating significance.
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Results |
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Vanadate-Stimulated eNO Production
Endothelial cells were incubated with the protein phosphotyrosine phosphatase inhibitor14 sodium orthovanadate, and the rate of eNO production was measured during continuous exposure to either Bk or calcium ionophore A23187. A small, continuous basal level of eNO was produced in the absence of vanadate, Bk, or A23187. Bk stimulated eNO production to a level about double that of the untreated controls (Figure 1A
), whereas A23187, which acts in a
receptor-independent manner to directly raise the intracellular calcium
concentration, caused a 4- to 5-fold increase in NO production
(Figure 1B). Vanadate alone stimulated the formation of eNO in a
time-dependent manner even in the absence of added Bk or A23187. When
Bk or A23187 was added to endothelial cells in the
presence of vanadate, however, there was a dramatic and sustained
enhancement of eNO formation to levels 10 to 20 times that of controls
(Figure 1). The amount of eNO generated by treatment with a
combination of vanadate and either Bk or A23187 was >2-fold greater
than the sum of eNO generated by treatment with the individual
components, indicating a synergistic effect of vanadate and Bk or
A23187 (Figure 1, summation curves).
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; vanadate, 
; Bk/vanadate and A23187/vanadate, 
; summation of
Bk and vanadate or A23187 and vanadate, +.
We also examined the effect of phosphotyrosine phosphatase inhibition
by vanadate over a range of Bk or A23187 concentrations. When cells
were incubated with Bk (Figure 2A), the
enhancing effects of 50 µmol/L vanadate were most apparent at
the lower concentrations and reached a plateau as Bk activation peaked
at 10 µmol/L. Incubation of endothelial cells
with A23187 led to a rapid and sustained rise in eNOS activity due to
calcium influx, and vanadate further stimulated eNO production.
This enhancement was most striking at A23187 concentrations <5
µmol/L, but enhancement of eNO production by vanadate
occurred even under conditions of apparent calcium saturation (Figure 2B). Incubation of cells in medium lacking calcium or containing
EGTA completely abolished the sustained generation of eNO, indicating
the necessity of a continuous influx of calcium from outside the cells
for extended eNOS activity. Likewise, removal of L-arginine
from the medium prevented the sustained generation of eNO (data not
shown).
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Effects of Protein Kinase and Other Inhibitors on
Vanadate-Stimulated NO Production
The specific involvement of tyrosine
phosphorylation in the enhancement of eNO
production is shown in Figure 3. Cells were pretreated with the
protein tyrosine kinase inhibitors genistein18
or tyrphostin ST63819 before activation with either Bk
(Figure 3A) or A23187 (Figure 3B), and the effects of
vanadate were abrogated. Phenylarsine oxide, which inhibits a class of
phosphotyrosine phosphatases that contain vicinal sulfhydryls at the
active site,20 in contrast to vanadate, which mimics the
transition state of phosphotyrosine phosphatase–catalyzed
reactions,21 did not cause enhancement of eNO
generation (Figure 3A), indicating that the class of
phosphotyrosine phosphatases inhibited by phenylarsine oxide was not
involved in the regulation of eNO production under these
conditions. Pretreatment of endothelial cells with the
NOS inhibitor L-NAME22 blocked eNO
production, whereas cotreatment with dexamethasone,
which blocks induction of the inducible isoform of NOS,23
had little effect (Figure 3B). Treatment with the protein
synthesis inhibitor cycloheximide did not alter the
vanadate response over the first 2 hours, suggesting that the
enhancement most likely involved a constitutive protein (data not
shown). Okadaic acid, a potent inhibitor of the
phosphoserine/phosphothreonine phosphatases pp1 and
pp2a,24 reduced the vanadate enhancement of eNO
production in Bk-treated cells (Figure 3A). When
endothelial cells were activated by A23187 in
the presence of okadaic acid, however, there was no effect on the
vanadate response, indicating that involvement of a putative serine
kinase was specific for the Bk-mediated signaling pathway (Figure 3B). The same basic experiments were repeated with primary
cultures of BAECs and human umbilical vein endothelial
cells, with essentially the same results (data not shown).
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Effect of Vanadate on eNOS Compartmentation and Expression
To determine whether vanadate affects the intracellular location
of eNOS, the relative levels of eNOS protein were measured in cell
lysates and subcellular fractions by immunoblotting and
densitometry. After centrifugation of
endothelial cell lysates at 100 000g,
95% of total eNOS protein was associated with the particulate
fraction, and vanadate treatment, in the presence or absence of Bk,
caused little or no change in this distribution. Also, there was no
significant difference in total eNOS protein under the various
treatment conditions compared with controls. Treatment of
endothelial cells with Bk, vanadate, or vanadate/Bk had
no significant effect on the subcellular distribution of eNOS, as
indicated by isopycnic centrifugation of cell lysates
through a sucrose gradient under conditions designed to separate
caveolae from denser membrane fractions.16 Most of
the eNOS protein was associated with the caveolar peak fraction
(fraction 3) under all treatment conditions (Figure 4). This fraction corresponded to the
interface between 15% and 25% sucrose. Noncaveolar membrane fractions
were distributed in denser regions of the gradient (fractions 4 to
8).16 The presence of eNOS and caveolin-1 in the peak
fractions shown in Figure 4 was confirmed by Western blotting
(data not shown). Because the samples were loaded from the bottom of
the gradients, cytosolic and cytoskeletal proteins would remain in the
45% sucrose sample zone (fractions 8 to 10).
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Effect of Vanadate on eNOS Activity Measured In Vitro
To determine whether vanadate affected eNOS directly, lysates of
untreated endothelial cells were assayed for eNOS
activity in the presence or absence of vanadate. No effect of vanadate,
at a concentration of 1 mmol/L, was observed (data not shown),
indicating that vanadate had no direct effect on eNOS. Lysates prepared
from cells that had been incubated with vanadate or vanadate/Bk for 4
hours showed a slight increase in eNOS activity compared with controls
(31.2±2.4 versus 25.3±2.0 pmol citrulline produced per minute per
milligram protein), but this possible increase in eNOS activity
measured in vitro does not account for the striking increase in NO
production by endothelial cells after treatment
with vanadate/Bk.
Vanadate-Induced Changes in Tyrosine-Phosphorylated
Proteins
The state of tyrosine phosphorylation of eNOS and
other protein components after the various treatment conditions was
analyzed by Western blotting, after immunoprecipitation with
anti-eNOS, by using an antibody probe to protein tyrosine phosphate. A
tyrosine-phosphorylated protein with an apparent
molecular mass of 125 kDa, along with lesser amounts of
tyrosine-phosphorylated proteins with apparent
molecular masses of 105 and 75 kDa, appeared to be specifically
associated with eNOS after vanadate/Bk treatment, as indicated by
coimmunoprecipitation with anti-eNOS (Figure 5). The apparent molecular mass of
eNOS run under identical conditions was 140 kDa. Little or no
tyrosine phosphorylation of eNOS was observed under
these conditions.
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Possible Role of hsp90 on Vanadate Stimulation of eNO
Production
hsp90 is a molecular "chaperone" with a molecular mass of
90 kDa that functions, in part, to facilitate the folding of certain
signal-transducing proteins.25 26 hsp90 has been shown to
associate with and stimulate eNOS activity.27
Geldanamycin, an inhibitor of hsp90,25 26
inhibited the vanadate/Bk and vanadate/A23187 stimulation of eNO
production with an apparent IC50 of
6 µmol/L (data not shown). Direct involvement of hsp90 in the
enhancement of eNOS activity under these conditions was further
supported by the finding that an increased amount of hsp90
coimmunoprecipitated with eNOS after vanadate/Bk treatment (Figure 6).
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Discussion |
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Early studies with purified NOS showed that the enzyme could be a substrate for a number of protein kinases.28 Although the bulk of in vivo eNOS phosphorylation occurs on serine/threonine, there have been reports of tyrosine phosphorylation of eNOS itself.6 7 Garcia-Cardeña et al6 found that treating endothelial cells with high levels (1 mmol/L) of vanadate caused an increase in basal tyrosine phosphorylation of eNOS and reduced eNOS activity by
50% in an in vitro immunoprecipitation-complex assay. Our results,
in contrast, indicate that lower levels (50 µmol/L) of vanadate
enhanced the Bk- and calcium ionophore–mediated production of
eNO. This enhancement was apparently indirect, because there was no
change in eNOS activity measured in vitro and no tyrosine
phosphorylation of eNOS was observed. These apparently
contradictory results may be due to different experimental conditions.
We used a significantly lower concentration of vanadate (50
µmol/L versus 1 mmol/L) and focused on receptor-mediated
production of eNO. It is unlikely that vanadate at the low
concentration used in this study (50 µmol/L) could cause such an
enhancement of eNOS activity through a nonspecific cellular effect.
Venema et al8 also reported that binding of Bk to
endothelial cells did not elicit tyrosine
phosphorylation of eNOS, but rather of a 90-kDa ENAP-1,
which appeared to lead to the translocation of eNOS to a
detergent-insoluble fraction. The change in eNOS localization, however,
did not result in a change in enzyme activity. Inhibition of tyrosine
kinases blocked phosphorylation of ENAP-1 and also
prevented the translocation of eNOS. We did not observe an alteration
in the overall pattern of eNOS intracellular distribution under our
experimental conditions. It should be noted, however, that our results
represent a "steady-state" condition, because eNO
production was monitored over several hours under the various
conditions. Therefore, transient changes would not be detected in
phosphorylation states or in intracellular location
that have been reported by other investigators.2
We found that continuous treatment of endothelial cells
with Bk for up to 6 hours stimulated eNO production to about
twice the basal level, whereas cotreatment with vanadate caused a much
greater (20-fold) and synergistic agonist response. The response to
vanadate was abolished by the tyrosine kinase inhibitors
genistein18 and tyrphostin,19 thus
demonstrating the specific involvement of tyrosine
phosphorylation in this enhancement. We also observed a
synergistic enhancement by vanadate of A23187-mediated activation of
eNO production, suggesting that the observed vanadate effect
was not mediated by Bk.
The tyrosine phosphorylation–associated enhancement of eNO production cannot be explained through an increase in the relative amounts of eNOS because Western blots showed no significant change in the distribution or in total eNOS levels in either particulate or cytosolic fractions after vanadate treatment, consistent with the lack of effect of cycloheximide on enhancement of eNO production by vanadate. The observed vanadate enhancement appears to be indirect, because vanadate had no effect on the specific activity of eNOS when included in an NOS assay of lysates from untreated cells. There was also little difference in the specific activity of eNOS from lysates of cells treated with Bk, vanadate, or Bk/vanadate.
Activation of eNOS may occur within the plasma membrane subcompartments known as caveolae, which are cholesterol-rich structures consisting of a scaffolding protein, caveolin-1, and an array of receptors and signal transduction factors. Caveolin-1 overexpression was found to inhibit eNOS activity in lysates from COS-7 cells coexpressing eNOS, and the effect was reversed by addition of calmodulin.29 It has also been reported that eNOS undergoes a flow-mediated dissociation from caveolin and a reassociation with calcium-calmodulin,30 which could serve to potentiate eNOS activity through maintaining the active dimeric form of the enzyme.31 Other proteins have been shown to bind to and modify eNOS activity, including ENAP-1,8 the Bk B2 receptor,32 33 and hsp90.27 34 We found that a 125-kDa tyrosine-phosphorylated protein, and possibly 105- and 75-kDa tyrosine-phosphorylated proteins, were associated with eNOS after treatment with Bk/vanadate. It was previously reported that eNOS coimmunoprecipitated with hsp90.27 Binding of hsp90 is associated with stimulation of eNOS activity,27 and enhanced binding of hsp90 to eNOS also appears to be involved in the estrogen receptor–mediated activation of eNOS.34 We found that hsp90 was apparently involved in the vanadate/Bk stimulation of eNO production, on the basis of the inhibition of this stimulation by geldanamycin, a specific inhibitor of hsp90, and on the apparent increase in hsp90 that coimmunoprecipitated with eNOS after exposure to vanadate. It should be noted that hsp90 and ENAP-1 are of the same apparent size and may even be the same protein (R.C. Venema, personal communication, 2000). Thus, the increase in tyrosine phosphorylation of certain proteins appears to trigger a marked enhancement of the activation state of eNOS through association with specific proteins that are presumably involved in regulating eNOS activity in vivo.
Clearly, there are different pathways that may affect eNOS activity, depending on the relative types and levels of phosphorylation/dephosphorylation in a system. Through this regulated interaction of enzymes and accessory proteins, a fluid complex is formed that promotes efficient coupling of the components of the signaling pathway. Our results support the view that protein tyrosine phosphorylation plays a prominent role in the complex signaling network regulating eNOS activity in vivo. This tyrosine phosphorylation–mediated modulation of eNOS activity appears to be due, in large part, to association/dissociation of "modulator" proteins, rather than to a direct modification of tyrosine residues of eNOS.
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Acknowledgments |
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This work was supported by grant 9750222N from the American Heart Association (National Center) and grant 9950864V from the American Heart Association, Florida Affiliate. We thank Dr Richard C. Venema for generously sharing unpublished information from his laboratory.
Received March 15, 2000; accepted June 12, 2000.
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