Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the Kd of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.
heat shock protein 90 modulation; nitric oxide synthase regulation; calmodulin binding affinity; tryptophan fluorescence; electron paramagnetic resonance spin trapping
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NITRIC OXIDE (NO) is a gaseous free radical that possesses a broad array of biological functions (15). The biosynthesis of NO in cells is carried out by a family of NO synthases (NOSs). Three NOS isoforms have been identified as neuronal NOS (nNOS, type I), inducible NOS (iNOS, type II), and endothelial NOS (eNOS, type III) (16). They all catalyze a similar reaction in which L-arginine and oxygen are converted to NO and L-citrulline. Calmodulin (CaM), FAD, flavin mononucleotide (FMN), and tetrahydrobiopterin are the essential cofactors for NOS catalysis (9, 20). FAD, FMN, and tetrahydrobiopterin are integrated into NOS structure once the enzymes are expressed in cells. The binding affinity between CaM and NOSs varies among different isoforms (14). iNOS has a constantly bound CaM and displays continuous enzymatic activity. On the other hand, dynamic CaM binding occurs with nNOS and eNOS. The binding between CaM and these two isoforms depends on the concentrations of cytosolic free Ca2+. In resting cells in which intracellular Ca2+ levels are low, CaM does not bind with nNOS or eNOS, rendering enzyme inactive. With the rise of intracellular Ca2+, Ca2+/calmodulin (Ca2+/CaM) binds with nNOS and eNOS, leading to active enzymatic function.
Under physiological conditions, NO production is strictly modulated to accommodate the need of homeostasis because abnormality of NO formation causes disease (10). Because NO is highly diffusible and cannot be stored in intracellular compartments, modulation of NO production in cells is primarily through regulating NOS activity. Several mechanisms have been revealed in NOS regulation. These include protein-protein interaction, protein phosphorylation, and subcellular targeting, etc. (6, 17). Among them, regulation via protein-protein interaction appears to be particularly important and versatile. In addition to the well-characterized CaM, recent studies highlighted the roles of several other scaffolding proteins. For example, nNOS was found to interact with postsynaptic density protein PSD-95 and PSD-93 in neurons (3). nNOS also binds with a 10-kDa protein, and this binding causes enzymatic inhibition (12). In endothelial cells, eNOS is localized in caveolae through interacting with caveolin-1 (5). This interaction results in inhibition of eNOS activity and decrease in NO production. Heat shock protein 90 (HSP90), a molecular chaperone, was first reported to associate with eNOS and acted as an allosteric enhancer (7). Subsequent studies suggested that HSP90 also facilitates nNOS activation in nNOS-transfected cells (2). Our recent study with purified enzyme preparations showed that HSP90 directly augments NO synthesis from nNOS (19). However, these prior findings were all derived from the indirect measurements of NO coproduct L-citrulline or NO metabolite nitrite and nitrate. The key evidence directly demonstrating the effect of HSP90 on NO synthesis from NOS is lacking. In addition, although our (19) functional studies and those of others (8) previously suggested that the mechanism of HSP90 is associated with enhancement of CaM binding to nNOS, it remains important to define the effect of HSP90 on CaM binding affinity to nNOS in a quantitative manner.
In this study, electron paramagnetic resonance (EPR) spectroscopy was employed to directly measure NO generation from purified nNOS as well as from nNOS-transfected cells. We studied the effects of HSP90 on NO synthesis from nNOS in vitro and in intact cells. Moreover, the enhancing action of HSP90 on the binding affinity between CaM and nNOS has been quantitatively determined by tryptophan fluorescence-quenching measurements.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. Cell culture materials were obtained from GIBCO BRL (Gaithersburg, MD). Bovine HSP90 and CaM, both >95% pure, were purchased from Sigma Chemical (St. Louis, MO). 2',5'-ADP-Sepharose 4B was the product of Amersham Pharmacia Biotech (Piscataway, NJ). L-[15N]arginine (guanidino 15N, 99% pure) was purchased from Isotec. L-[14C]arginine was from NEN (Boston, MA). Diethyldithiocarbamate (DETC) was the product of Aldrich (Milwaukee, WI). A-23187, NADPH, L-arginine, tetrahydrobiopterin, geldanamycin (GA), NG-nitro-L-arginine methyl ester (L-NAME), and other reagents were purchased from Sigma unless otherwise indicated.
nNOS purification.
Recombinant rat nNOS was purified from the stably transfected human
embryonic kidney 293 cells (nNOS-HEK-293; a gift from Dr. Solomon H. Snyder at Johns Hopkins University School of Medicine) as previously
described (19). In brief, nNOS-transfected cells were
grown in minimum essential medium with 10% heat-inactivated fetal calf
serum. Cells were harvested and homogenized in buffer A that
contained 50 mM Tris · HCl, pH 7.4, 2 mM EDTA, 1 mM
dithiothreitol, and protease inhibitor cocktail tablets (Boehringer).
After being centrifuged (16,000 g, 10 min) at 4°C, the
supernatant was applied to a 2',5'-ADP-Sepharose 4B column
preequilibrated in buffer A. The column was extensively
washed with buffer A containing 450 mM NaCl and
Tris · HCl buffer (50 mM, pH 7.4). The protein was then eluted
with 10 mM NADPH in 50 mM Tris · HCl (pH 7.4). The eluate was
washed and concentrated using Centricon-100 (Amicon) concentrators.
Protein content of the preparations was assayed with the Bradford
reagent (Bio-Rad) using bovine serum albumin as standard. The purity of
nNOS was determined by SDS-PAGE and visualized with Coomassie brilliant
blue (R-250, Bio-Rad) staining. The purified nNOS preparations
exhibited one prominent band on gels with a molecular mass of 160 kDa.
Enzymatic assay by oxyhemoglobin showed that the activity of the
purified nNOS preparations was in the range of 600-800
nmol · mg1 · min
1 at 37°C
(13). Purified nNOS samples were stored in 50 mM
Tris · HCl (pH 7.4) buffer with 10% glycerol at
80°C.
EPR spectroscopy and spin trapping. An iron complex of DETC [Fe3+-DETC2, (Fe-DETC)] was employed to trap NO generated from nNOS (21, 22). DETC was dissolved in 20% fat emulsion (Liposyn III; Abbott Laboratories). Fe-DETC was prepared by adding ferrous ammonium sulfate [Fe(NH4)2(SO4)2 · 6H2O] solution to DETC solution with frequent shaking. Fresh preparations of Fe-DETC were used in each EPR measurement. NO measurements were conducted in 500 µl of buffer containing 50 mM Tris · HCl (pH 7.4), 0.5 mM NADPH, 100 µM L-[15N]arginine, 0.5 mM Ca2+, 0.2 µg/ml CaM, 1 µM tetrahydrobiopterin, 30 nM nNOS, and 0.5 mM Fe-DETC. In the experiments determining the effect of HSP90, nNOS and HSP90 were incubated for 15 min before measurements.
To detect NO generation in intact cells, EPR NO measurements were performed on nNOS-HEK-293 cells (107 cells/ml) in phosphate-buffered saline (with 1 mM Ca2+) (23). nNOS was activated by Ca2+ ionophore (A-23187, 1 µM), and NO release from the cells was trapped by Fe-DETC (0.5 mM). In the experiments examining the role of HSP90 in modulating NO synthesis in intact cells, the nNOS-HEK-293 cells were preincubated with the HSP90 inhibitor GA for 1 h before EPR measurements. EPR spectra were recorded in a quartz flat cell at room temperature (23°C) with a Bruker ER 300 spectrometer operating at X-band with a TM110 cavity using a modulation frequency of 100 kHz, modulation amplitude of 0.5 gauss, microwave power of 20 mW, and microwave frequency of 9.785 GHz. The microwave frequency and magnetic field were precisely measured using an EIP 575 microwave frequency counter and Bruker ER 035 nuclear magnetic resonance gaussmeter. NO signals were quantitated by double integration as previously described (23, 24).Fluorescence measurements of CaM binding.
Fluorescence spectra were acquired at room temperature (23°C) on a
Spex FluoroMax spectrofluorometer (Spex Industries). Tryptophan residues of nNOS were excited at 295 nm in 1 ml of buffer containing 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.1 mM
Ca2+, and the emission spectra were recorded from 280 to
420 nm (18). nNOS (25 nM) was titrated with CaM (1-50
nM) in the presence and absence of HSP90 (50 nM). Changes in the final
volume were <1%. Emission values were corrected by the background
spectrum recorded from the buffer under matched experimental
conditions. The amounts of nNOS bound with CaM were calculated from the
fractional fluorescence quenching at 338 nm using a similar method
described by Ehlers et al. (4). The concentrations of
bound nNOS, bound CaM, free nNOS, and free CaM were used to construct
saturation curves and Scatchard plots. Lines were fit to the data using
the single-site Scatchard relationship B/F = B/Kd + Bmax/Kd, where B is the amount bound, F is the amount free, Kd is the
dissociation constant, and Bmax is the maximal number of
binding sites. Kd was calculated using the
regression of saturation curve program (PRISM, GraphPad Software).
Western blotting. Control and GA-treated cells were lysed in a boiling SDS-sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 40 mM dithiothreitol, and 10% glycerol). After a brief centrifugation (16,000 g, 5 min), supernatants were loaded on 4-20% polyacrylamide gels and subjected to electrophoresis. Migrated proteins on gels were then transferred to a nitrocellulose membrane and probed with an anti-nNOS monoclonal or anti-HSP90 antibody (1:4,000 dilution, Transduction Laboratories). Immunoblots were developed on films using an enhanced chemiluminescence technique (ECL, Amersham) (25).
Statistics. Data are expressed as means ± SE. Comparisons were made using a two-tailed paired or unpaired Student's t-test. Differences were considered to be statistically significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To directly determine the effect of HSP90 on NO synthesis from
nNOS, we performed EPR measurements of NO using the spin trap Fe-DETC.
To ensure that only the NO generated by nNOS was measured, L-[15N]arginine (15N labeling of
guanidino groups) was used as enzyme substrate. In the absence of
enzyme, no signal was detected from the reaction system containing
L-[15N]arginine and nNOS cofactors (Fig.
1A). After adding nNOS
(30 nM), prominent EPR signals were seen (Fig. 1B).
These signals displayed the characteristic doublet spectrum of
15NO-Fe-DETC. These 15NO signals were totally
abolished by the NOS inhibitor L-NAME (1 mM; Fig.
1C), reconfirming that the measured 15NO was
synthesized by nNOS. To determine the effect of HSP90, nNOS was
preincubated with different concentrations of HSP90. As shown in Fig.
1, D-F, HSP90 increased 15NO production
from nNOS in a dose-dependent manner. With the presence of 1.5 µM
HSP90, 15NO production was increased more than twofold
(316.6 ± 51.4% of control, P < 0.001; Fig.
2A). This increase of NO
production was blunted by the specific HSP90 inhibitor GA (20 µM;
Fig. 1G). Moreover, the irrelevant bovine serum albumin had
no effect on NO generation from nNOS at the identical concentration of
HSP90 (1.5 µM; Fig. 1H). We also studied the effect of
HSP90 on the time course of NO production by nNOS. As displayed in Fig.
2B, NO production from nNOS increased as a function of time,
and this was totally blocked by L-NAME. HSP90 dose
dependently increased the magnitude of NO production while the time
course of NO formation by nNOS appeared to be unchanged.
|
|
To quantitate the effect of HSP90 on CaM binding affinity to nNOS, we
determined the binding kinetics of CaM to nNOS by measuring the
fluorescence quenching of tryptophan residues in nNOS. With an
excitation wavelength at 295 nm, the tryptophan residues in nNOS
exhibited a maximal emission at 338 nm. Adding CaM dose dependently quenched the tryptophan fluorescence of nNOS (Fig.
3A, left). Measurements of the fractional fluorescence quenching allowed a
Scatchard analysis of the binding between CaM and nNOS. This analysis
revealed a linear Scatchard plot, indicating a single CaM binding site
on nNOS (Fig. 3A, right). The calculated
Kd of CaM to nNOS was 9.4 ± 1.8 nM. In the
presence of HSP90 (50 nM), CaM caused a much more dramatic quenching of
tryptophan fluorescence compared with that in the absence of HSP90
(Fig. 3B, left). The maximal emission wavelength
was blue shifted to ~328 nm, indicating that the tryptophan residues
in nNOS were in a more hydrophobic environment. Scatchard analysis
showed the Kd of CaM to nNOS was significantly
reduced while HSP90 was present (0.5 ± 0.1 nM, P < 0.01, compared with the Kd in the absence of
HSP90). These data provided quantitative evidence demonstrating that
HSP90 enhances CaM binding affinity to nNOS.
|
To determine the role of HSP90 in regulating NO synthesis in intact
cells, EPR NO measurements were performed on nNOS-HEK-293 cells. As
shown in Fig. 4A, activation
of nNOS by the Ca2+ ionophore A-23187 (1 µM) resulted in
strong EPR signals from cells (Control). These signals exhibited
triplet 14NO spectrum because of the natural abundant
L-[14N]arginine in the cytosol. The addition
of L-NAME (5 mM) abolished the NO signals
(L-NAME), confirming that the NO generation was derived
from nNOS. To explore the role of HSP90, cells were treated with the
HSP90 inhibitor GA (20 µM) for 1 h. GA treatment caused a
significant decrease of NO signals from cells (GA). Quantitation of
these NO signals showed that NO production from GA-treated cells was
inhibited to 47.9 ± 4.1% of that from control cells (P < 0.001; Fig. 4B). To determine whether
decreased NO production was due to the reduction of cytosolic nNOS or
HSP90 content, we compared the nNOS and HSP90 protein levels in control
and GA-treated cells. As shown in Fig. 4C, the same amounts
of nNOS and HSP90 protein were found in control and GA-treated cells.
Thus inhibition of HSP90 decreased NO production from nNOS, and this
was not mediated by the reduction of intracellular nNOS content.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of this study is to determine the role of HSP90 in regulating nNOS function by direct NO measurement. While a number of methods have been used in NO measurement, most of them are based on the detection of NO metabolites or coproduct (1). The most commonly used assay is the measurement of nitrite and nitrate by Griess reaction (2, 11). Although measurement of nitrite and nitrate offers a simple way to indirectly reflect NO formation, the specificity of this assay has been a major concern because nitrite and nitrate may arise from a number of sources in the biological system (1). The detection of NO coproduct L-citrulline is indeed specific, but it does not afford a direct visualization of NO generation from NOS. NO is a paramagnetic free radical with an unpaired electron. So far, EPR spectroscopy remains the most unambiguous technique in free radical detection. With the spin trap Fe-DETC, we directly measured NO production from nNOS. To eliminate the possible contamination of NO generated from other sources such as reduction of nitrite (26), we used L-[15N]arginine instead of nature-abundant 14N L-arginine. The unique doublet EPR spectrum of 15NO warranted that only NO derived from the L-arginine-NOS pathway was measured. By using these techniques, we found that HSP90 dose dependently increased 15NO signals from nNOS. This is unequivocal evidence directly proving that HSP90 enhances NO synthesis from nNOS.
Our previous studies showed that HSP90 shifts the CaM-nNOS dose-response curve to the left, suggesting that HSP90 enhances the binding between CaM and nNOS (19). This assertion was also supported by in vitro protein binding assay. In the present study, we sought to quantitatively define the effect of HSP90 on CaM binding affinity to nNOS. Upon the binding of CaM, a change of nNOS conformation occurs, leading to the quenching of tryptophan fluorescence. Measurements of tryptophan fluorescence quenching have been utilized to study the binding between CaM and nNOS (18). Using this method, we measured the Kd of CaM to nNOS to be 9.4 nM in the absence of HSP90. This value is well in agreement with the EC50 of CaM (10.2 nM) obtained from enzymatic assay (9, 19). In the presence of HSP90, the Kd value of CaM was markedly decreased to 0.5 nM, indicating an increased affinity between CaM and nNOS. Thus these quantitative binding analyses supported the previous results from functional studies and confirmed that HSP90 enhances CaM binding to nNOS.
The role of HSP90 in modulating NO synthesis from nNOS in intact cells has been indirectly assessed by Bender et al. (2). With similar nNOS-transfected cells, these researchers showed that nitrite and nitrate release was reduced in GA-treated cells. In their study, cells were exposed to GA for 6 h, and the reduction of NO production may be partially due to decreased cytosolic nNOS content. Indeed, they reported that intracellular nNOS protein levels decreased by 50% after a 24-h GA treatment. To definitively establish the role of HSP90 in nNOS regulation in cells, direct NO measurements need to be conducted on the cells with equal amounts of nNOS. In the present study, NO production from cells was directly trapped by Fe-DETC. The cells were subjected to a 1-h GA treatment to block HSP90. Western blotting confirmed that this treatment did not alter cytosolic nNOS protein levels. We found that HSP90 blockade decreased NO production from nNOS >50%. These results indicated that HSP90 functions as an allosteric enhancer for nNOS in cells. Loss of the interaction with HSP90 decreases nNOS activity despite the presence of the same amounts of enzyme in the cytosol.
In summary, we have provided direct evidence demonstrating that HSP90 enhances NO synthesis from nNOS, and this action is mediated by the enhancement of CaM binding to nNOS. Coupling with HSP90 is, therefore, important for normal nNOS function. Whether disruption of HSP90/nNOS coupling plays a role in the mechanism of diseases associated with decreased NO production should be an interesting area for future study.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Alexandre Samouilov for help in preparing Fe-DETC.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants AG-00835 (to Y. Xia) and HL-63744 (to J. L. Zweier) and Grant-in-Aid awards from the American Heart Association (to Y. Xia).
Address for reprint requests and other correspondence: Y. Xia, Johns Hopkins Asthma Center, Rm. LA-14, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: yongxia{at}jhmi.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 June 2001; accepted in final form 7 August 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Archer, S.
Measurement of nitric oxide in biological models.
FASEB J
7:
349-360,
1993
2.
Bender, AT,
Silverstein AM,
Demady DR,
Kanelakis KC,
Noguchi S,
Pratt WB,
and
Osawa Y.
Neuronal nitric-oxide synthase is regulated by the Hsp90-based chaperone system in vivo.
J Biol Chem
274:
1472-1478,
1999
3.
Brenman, JE,
Chao DS,
Gee SH,
McGee AW,
Craven SE,
Santillano DR,
Wu Z,
Huang F,
Xia H,
Peters MF,
Froehner SC,
and
Bredt DS.
Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and -syntrophin mediated by PDZ domains.
Cell
84:
757-767,
1996[ISI][Medline].
4.
Ehlers, MD,
Zhang S,
Bernhardt JP,
and
Huganir RL.
Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit.
Cell
84:
745-755,
1996[ISI][Medline].
5.
Feron, O,
Belhassen L,
Kobzik L,
Smith TW,
Kelly RA,
and
Michel T.
Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells.
J Biol Chem
271:
22810-22814,
1996
6.
Fleming, I,
and
Busse R.
Signal transduction of eNOS activation.
Cardiovasc Res
43:
532-541,
1999[ISI][Medline].
7.
García-Cardeña, G,
Fan R,
Shah V,
Sorrentino R,
Cirino G,
Papapetropoulos A,
and
Sessa WC.
Dynamic activation of endothelial nitric oxide synthase by Hsp90.
Nature
392:
821-824,
1998[ISI][Medline].
8.
Gratton, JP,
Fontana J,
O'Connor D,
García-Cardeña G,
McCabe TJ,
and
Sessa WC.
Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1.
J Biol Chem
275:
22268-22272,
2000
9.
Griffith, OW,
and
Stuehr DJ.
Nitric oxide synthases: properties and catalytic mechanism.
Annu Rev Physiol
57:
707-736,
1995[ISI][Medline].
10.
Gross, SS,
and
Wolin MS.
Nitric oxide: pathophysiological mechanisms.
Annu Rev Physiol
57:
737-769,
1995[ISI][Medline].
11.
Ignarro, LJ,
Buga GM,
Wood KS,
Byrns RE,
and
Chaudhuri G.
Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
Proc Natl Acad Sci USA
84:
9265-9269,
1987[Abstract].
12.
Jaffrey, SR,
and
Snyder SH.
PIN: an associated protein inhibitor of neuronal nitric oxide synthase.
Science
274:
774-777,
1996
13.
Kelm, M,
Feelich M,
Spahr R,
Piper HM,
Noack E,
and
Schrader J.
Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells.
Biochem Biophys Res Commun
154:
236-244,
1988[ISI][Medline].
14.
Marletta, MA.
Nitric oxide synthases: aspects concerning structure and catalysis.
Cell
78:
927-930,
1994[ISI][Medline].
15.
Moncada, S,
Palmer RM,
and
Higgs EA.
Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol Rev
43:
109-142,
1991[ISI][Medline].
16.
Nathan, C,
and
Xie Q-W.
Nitric oxide synthases: roles, tolls, and controls.
Cell
78:
915-918,
1994[ISI][Medline].
17.
Papapetropoulos, A,
Rudic RD,
and
Sessa WC.
Molecular control of nitric oxide synthases in the cardiovascular system.
Cardiovasc Res
43:
509-520,
1999[ISI][Medline].
18.
Sheta, EA,
McMillan K,
and
Masters BSS
Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase.
J Biol Chem
269:
15147-15153,
1994
19.
Song, Y,
Zweier JL,
and
Xia Y.
Heat-shock protein 90 augments neuronal nitric oxide activity by enhancing Ca2+/calmodulin binding.
Biochem J
355:
357-360,
2001[ISI][Medline].
20.
Stuehr, DJ.
Structure-function aspects in the nitric oxide synthases.
Annu Rev Pharmacol Toxicol
37:
339-359,
1997[ISI][Medline].
21.
Vanin, AF.
Iron diethyldithiocarbamate as a spin trap for nitric oxide detection.
Methods Enzymol
301:
269-279,
1999[ISI][Medline].
22.
Vanin, AF,
Liu X,
Samouilov A,
Stukan RA,
and
Zweier JL.
Redox properties of iron-dithiocarbamates and their nitrosyl derivatives. Implications for their use as traps of nitric oxide in biological systems.
Biochim Biophys Acta
1474:
365-377,
2000[ISI][Medline].
23.
Xia, Y,
Dawson VL,
Dawson TD,
Snyder SH,
and
Zweier JL.
Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.
Proc Natl Acad Sci USA
93:
6770-6774,
1996
24.
Xia, Y,
and
Zweier JL.
Direct measurement of nitric oxide generation from nitric oxide synthase.
Proc Natl Acad Sci USA
94:
12705-12710,
1997
25.
Xia, Y,
and
Zweier JL.
Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages.
Proc Natl Acad Sci USA
94:
6954-6958,
1997
26.
Zweier, JL,
Wang P,
Samouilov A,
and
Kuppusamy P.
Enzyme-independent formation of nitric oxide in biological tissues.
Nat Med
1:
804-809,
1995[ISI][Medline].