(Circulation. 2001;103:2188.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Effects of In Vivo Nitroglycerin Treatment on Activity and Expression of the Guanylyl Cyclase and cGMP-Dependent Protein Kinase and Their Downstream Target Vasodilator-Stimulated Phosphoprotein in Aorta
From University Hospital Eppendorf, Division of Cardiology, Hamburg (M.O., H.M., A.W., U.H., T. Meinertz, T. Münzel); Institute of Cardiovascular Physiology, J.W. Goethe-University Clinics, Frankfurt/Main (A.M., S.K., A.T.); Bayer AG, Wuppertal (J.-P.S.); and Institut für Klinische Biochemie und Pathobiochemie (A.S., U.W.), Würzburg, Germany.
Correspondence to Thomas Münzel, MD, Universitätskrankenhaus Eppendorf, Abteilung für Kardiologie, Martinistraße 52, 20246 Hamburg, Germany. E-mail muenzel{at}uke.uni-hamburg.de
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well.
Methods and Results—We
therefore studied the effects of 3-day NTG treatment of rats and
rabbits on activity and expression of the immediate NO target soluble
guanylyl cyclase (sGC) and on the cGMP-activated protein kinase
I (cGK-I). Tolerance was induced either by chronic NTG infusion via
osmotic minipumps (rats) or by NTG patches (rabbits). Western blot
analysis, semiquantitative reverse transcription–polymerase
chain reaction, and Northern blot analysis revealed significant
and comparable increases in the expression of sGC
1 and ß1 subunit
protein and mRNA. Studies with the oxidative fluorescent dye
hydroethidine revealed an increase in superoxide in the
endothelium and smooth muscle. Stimulation with NADH
increased superoxide signals in both layers. Although cGK-I expression
in response to low-dose NTG was not changed, a strong reduction in
vasodilator-stimulated phosphoprotein (VASP) serine239
phosphorylation (specific substrate of cGK-I) was
observed in tolerant tissue from rats and rabbits. Concomitant in vivo
and in vitro treatment with vitamin C improved tolerance, reduced
oxidative stress, and improved P-VASP.
Conclusions—We therefore conclude that increased expression of sGC in the setting of tolerance reflects a chronic inhibition rather than an induction of the sGC–cGK-I pathway and may be mediated at least in part by increased vascular superoxide.
Key Words: protein kinases • vasodilator-stimulated phosphoprotein • nitrate tolerance • hydroethidine • superoxide production
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Nitroglycerin (NTG) induces vasorelaxation by releasing nitric oxide (NO).1 NO, an endothelium-derived relaxing factor (EDRF), activates the target enzyme soluble guanylyl cyclase (sGC) and increases tissue levels of the second messenger cGMP.2 cGMP activates a cGMP-dependent protein kinase (cGK) that mediates vasorelaxation via phosphorylation of proteins that regulate intracellular Ca2+ levels.3 Although short-term application of NTG exhibits high vasodilator and anti-ischemic efficacy, this activity is rapidly lost on long-term treatment because of the development of tolerance.4 The mechanisms underlying this phenomenon are likely to be multifactorial and may involve neurohormonal adjustments as well as changes intrinsic to the vasculature itself, such as desensitization of the sGC5 and/or decreased expression of the cGMP-dependent kinase I (cGK-I).6 Using lucigenin-derived chemiluminescence, we recently showed that NTG increases vascular superoxide in endothelial but also in smooth muscle cells.4 Increased vascular superoxide may inactivate NO formed via NTG biotransformation but may also have untoward effects on the downstream NO targets sGC and cGK-I .
The hemoprotein sGC is the predominant intracellular NO
receptor in vascular smooth muscle cells (for review see Reference 77 ).
The active enzyme is an obligate heterodimer, which in most mammalian
tissues consists of 1 (76- to 82-kDa) and
ß1 (70-kDa) protein
subunits.7 Chronic in vivo
NTG treatment attenuates the increase in vascular cGMP levels in
response to acute challenge with endothelium-derived NO
and other nitrovasodilators5
because of so-called cross-tolerance. Furthermore, in vitro exposure of
cells and tissues with high NO concentrations desensitizes sGC to
nitrovasodilators, a phenomenon called in vitro
tolerance.8 This led to
speculations that sGC desensitization may also occur in in vivo
tolerance.
The cGK-I is highly expressed in vascular smooth muscle cells.9 Studies with cGK-I–deficient mice and human cells demonstrated a complete disruption of the NO/cGMP signaling pathway in vascular tissue.10 Therefore, the activity and/or expression of cGK-I may critically influence NTG-induced vasorelaxation. The vasodilator-stimulated phosphoprotein (VASP) phosphorylation and in particular VASP serine239 phosphorylation (P-VASP) has been shown to be a useful monitor for cGK-I activity in intact cells.11 VASP, a protein highly expressed in vascular cells, including platelets, endothelial cells, and vascular smooth muscle cells, is phosphorylated at 3 distinct sites (serine157, serine239, and threonine278) by both cGK-I and cAMP-dependent protein kinases with overlapping specificity and efficiency.11 Experiments with cGK-I–deficient human endothelial cells and platelets established that NO donor– and cGMP-mediated VASP phosphorylation is mediated by the cGK-I.12 13 On the basis of this evidence, the present study was designed to examine the effects of in vivo NTG tolerance on sGC expression, as well as on downstream targets of sGC, cGK-I, and its substrate VASP. The oxidative fluorescent dye hydroethidine was used to determine the precise localization (endothelium versus smooth muscle) of superoxide production.14 To elucidate a role for superoxide in modulating P-VASP, animals were treated concomitantly with the antioxidant vitamin C.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animal Model, In Vivo Nitrate Tolerance
Male Wistar rats (weight 250 to 300 g) and New Zealand White rabbits (weight 3 to 4 kg) were studied. Tolerance was induced with miniosmotic Alzet pumps15 or NTG patches4 (release rate 0.5 mg/h), leading to NTG delivery rates of
10 and 3.0 µg ·
kg-1 ·
min-1, respectively). Because high
concentrations of NO donors have been shown to decrease the expression
of cGK-I,6 NTG was infused in
rats in low (10 µg · kg-1 ·
min-1) and high (100 µg ·
kg-1 ·
min-1) concentrations. To test the effects
of oxidative stress on P-VASP, rabbits were treated for 3 days with the
antioxidant vitamin C (500 mg/d PO), which was mixed into the drinking
water.
Vessel Preparation and Organ Chamber
Studies
Aortic rings were suspended in organ chambers, and
the NTG dose-response relationship with and without vitamin C (1
mmol/L; 30 minutes) was established as
described.4
Determination of Vascular Superoxide
Production
Lucigenin Assay
Measurement of vascular superoxide was performed with
lucigenin (5 µmol/L) as
described.16 To test the
effects of in vitro incubations on vascular superoxide
production, aortic rings from NTG-treated rabbits were
incubated with vitamin C (10 mmol/L; 60
minutes).
Oxidative Fluorescent
Microtopography
The oxidative fluorescent dye hydroethidine
was used to evaluate the in situ concentration of superoxide as
described.14 In the presence
of superoxide, hydroethidine is rapidly oxidized to ethidium bromide
(EtBr), where it is trapped by intercalation with DNA. Some rings were
incubated with NADH (100 µmol/L; 30 minutes) before being cross
sectioned.
RT-PCR of 1 and
ß1 sGC mRNA
RNA extraction from aortic tissue, quantification,
and reverse transcription–polymerase chain reaction (RT-PCR) for both
sGC subunits and elongation factor II were performed as
described.17
RNA Blot Hybridization for
1 and ß1 sGC
mRNA
Total aortic RNA was extracted as described above,
modified by the single-step RNA isolation method with Trizol reagent
(Gibco-BRL). Poly(A)+ RNA was extracted from
2 mg of total RNA with oligo(dT) cellulose (Gibco-BRL). One to 2 µg
of poly(A)+ mRNA was fractionated in 1.2%
agarose-formaldehyde gels containing EtBr. The RNA was
transferred to Porablot nylon membranes (Macherey-Nagel), cross-linked
by exposure to UV light, and baked (80°C) for 2 hours. Membranes were
hybridized with biotinylated cDNA probes (BioPrime DNA labeling system,
Gibco-BRL) recognizing rat sGC 1 and
ß1 subunits and elongation factor II, the
latter being used for verification of equal loading. Positive bands
were identified with the Photogene nucleic acid detection system
(Gibco-BRL).
sGC Western Blot
Western blotting for sGC 1
and ß1 subunits was performed as described
previously for the ß1
subunit.17
Determination of sGC Activity
The activity of sGC was determined by conversion of
[-32P]GTP into
[32P]cGMP as
described.8
Detection of cGK-I Expression, cGK-I Activity,
and P-VASP
Aortic tissue was frozen and homogenized
in liquid nitrogen. SDS-PAGE and electroblotting was performed as with
sGC. The membrane was then divided horizontally at 65 kDa. A polyclonal
antibody against cGK-I13 and
a mouse monoclonal antibody (16C2) specific for VASP
phosphorylated at
serine23911 was used. All
data reported here as P-VASP indicate VASP
phosphorylation at serine239. A second blot was used to
detect VASP expression by use of a
phosphorylation-independent monoclonal antibody against
VASP (IE273).18
Immunodetections were done with anti-rabbit/mouse secondary antibodies
and were visualized as described above. As positive controls for cGK-I
or P-VASP, we used 10 ng cGK-I and 10 µg protein of sodium
nitroprusside (SNP)–stimulated human
platelets,11
respectively.
In separate experiments, aortas from control and NTG-treated rats were incubated with high concentrations of SNP (10 µmol/L), which have been shown to cause maximal relaxation in control and tolerant aortas. This was performed to test whether the cGMP-dependent signaling pathway is still intact in tolerant tissue. To study a role for oxidative stress in the regulation of P-VASP in response to NTG treatment, aortas from tolerant rabbits were treated in vitro with vitamin C (1 mmol/L for 30 minutes).
Statistical Analysis
Results are expressed as mean±SEM. The
ED50 value for each experiment was obtained by
logit transformation. To compare superoxide production, P-VASP
and VASP, cGK-I, sGC expression, and sGC activity in normal and
NTG-treated animals, 1-way ANOVA was used. Comparisons of vascular
responses were performed with multivariate ANOVA. A
Scheffé’s post hoc test was used to examine differences between
groups when significance was indicated. Probability values of
P<0.05 were considered
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of NTG Treatment on EtBr Fluorescence in Endothelial and Smooth Muscle Cells
NTG treatment increased EtBr fluorescence in the endothelium and smooth muscle, reflecting increased superoxide. Preincubation of aortic segments with NADH markedly increased EtBr fluorescence in the endothelium and media of NTG-treated animals but not in controls (Figure 1
).
|
Effects of In Vivo NTG Treatment on sGC
1 and ß1 mRNA and
Protein Expression in Rat Aorta
Compared with controls, NTG treatment caused a 2-fold
increase in the mRNA abundance of sGC 1 and
ß1 as assessed by semiquantitative RT-PCR
(Figure 2A) and by nonradioactive Northern blot technique
(Figure 2B) (n=4;
P<0.05). The increase in sGC
message was accompanied by a comparable increase in sGC
1 and ß1 protein in
rat aorta
(Figure 3) as well as in rabbit aorta (data not
shown).
|
|
Effects of In Vivo NTG Treatment on sGC
Activity in Homogenates From Rat Aorta
The SNP-stimulated sGC activity in protein extracts
from tolerant aortas was higher than in controls
(Figure 4).
|
Influence of In Vivo NTG Treatment on cGK-I
Expression and on the Levels of Basal and Ex Vivo SNP-Induced
P-VASP
In vivo NTG treatment caused a substantial drop in
P-VASP in rat
(Figure 5) as well as in rabbit aorta
(Figure 6) without altering cGK-I and VASP expression.
Incubation of control and tolerant tissue with high concentrations of
SNP caused comparable maximal levels of P-VASP in rat aorta
(Figure 5). ß-Tubulin expression did not change in response
to NTG treatment
(Figure 6).
|
|
Influence of In Vivo Low- and High-Dose NTG
Treatment on Vascular cGK-I Expression
Infusion of NTG in high (100 µg ·
kg-1 ·
min-1) but not low (10 µg ·
kg-1 ·
min-1) concentrations significantly
decreased cGK-I expression in rat aorta
(Figure 7).
|
Influence of In Vitro and In Vivo Vitamin C
Treatment on NTG Dose-Response Relationship, Vascular Superoxide, and
P-VASP
In vitro vitamin C treatment of aortas from in vivo
NTG-treated rabbits improved tolerance, reduced vascular superoxide,
and increased P-VASP. The effects were even more striking in New
Zealand White rabbits treated concomitantly with vitamin C in vivo
(Figure 8).
|
P<0.05 vs NTG-treated. |
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We demonstrate here for the first time that in vivo treatment with NTG leads to increased expression of the NO target enzyme sGC in 2 different animal models of nitrate tolerance. Downstream signaling of sGC by cGMP-dependent protein kinase (cGK-I), however, was markedly attenuated, as indicated by the pronounced reduction in the basal phosphorylation of the cGK-I substrate VASP. The oxidative fluorescent probe hydroethidine showed increased superoxide in endothelial as well as in smooth muscle cells, which was further increased after stimulation with NADH. Tolerant tissue responded fully to the nitrovasodilator SNP in concentrations that cause maximal relaxation in control and tolerant tissue, with comparable maxima in P-VASP. In vivo as well as in vitro treatment with the antioxidant vitamin C improved vascular relaxations to NTG in NTG-treated rabbits, reduced vascular superoxide, and increased P-VASP, indicating a contribution of oxidative stress to these phenomena. Decreased P-VASP in the presence of increased expression of sGC may indicate that in the setting of tolerance, the NO/cGMP pathway is inhibited rather than activated.
Previously, we and others have shown that treatment of rats and rabbits with NTG leads to a marked attenuation of vasodilator responses to NTG as well as to NO/EDRF-eliciting agonists.4 5 Because in vivo NTG tolerance is associated with decreased basal and NO-induced cGMP level in vascular tissue, Molina et al5 hypothesized that sGC in vascular smooth muscle was desensitized by chronic exposure to NTG. Recent studies have shown that chronic NTG treatment increases superoxide in endothelial as well as in smooth muscle cells4 15 and that removal of endothelium as well as treatment with liposomal superoxide dismutase (SOD) partially but not completely improved NTG-elicited relaxations in tolerant tissue.4 This observation suggests that increased endothelial and/or smooth muscle superoxide production may be an important determinant of in vivo nitrate tolerance.
Effects of Chronic NTG Treatment on sGC
Activity and Expression
By analyzing the effects of 3-day infusion of rats with
NTG on sGC activity in the cytosolic fraction from
endothelium-intact aortic tissue
homogenate, we made the surprising observation that the
SNP-stimulated sGC activity in tolerant aorta was significantly higher
than in nontolerant aorta. In direct support of these findings,
activation by the NO-independent sGC activator YC-1 (100
µmol/L)19 was also 2-fold
higher in tolerant aortic cytosol than in control cytosol (data not
shown). The fact that vascular sGC, although "desensitized" in
intact tissue, is fully responsive to NO in protein extracts from
tolerant tissue suggests that the desensitizing process in intact
tissue is not operative in protein extracts. This is in accordance with
the hypothesis that in intact tissue, superoxide generated by a
membrane-bound NADH oxidase accounts for the reduced bioavailability of
NTG-derived NO and may also directly inhibit
sGC.19 This oxidase is not
active in the protein extract under the present sGC assay
conditions, and consequently, activation of sGC by NO is not
impaired.
By analyzing the effects of NTG treatment on the expression of sGC in tolerant rat and rabbit aorta, we found a significant increase in sGC subunit mRNAs and protein. At first glance, the increased sGC expression in NTG tolerance is unexpected. Recently, however, endothelial dysfunction of the aorta in an animal model of chronic ischemic cardiomyopathy was found to be associated with increased vascular superoxide production, depressed vascular cGMP levels, and increased expression of NO synthase III and the sGC ß1 subunit.20 Because cGMP responses were restored by the SOD mimetic Tiron, the authors concluded that the sGC under these conditions was chronically inhibited by increased vascular superoxide production. The coincidence between the 2 animal models (heart failure and nitrate tolerance) with respect to sGC expression is striking and may point to a common mechanism of upregulation of sGC expression.
Effects of Chronic NTG Treatment on the
Expression and Activity of cGK-I
To assess whether the downstream target of sGC, cGK-I,
was also affected by NTG, we analyzed the expression of this
protein by Western blotting. In aortas from NTG-tolerant rats and
rabbits, no changes in cGK-I expression were observed. These data
appear to be inconsistent with the recent findings from Soff et
al,6 who reported significant
downregulation of cGK-I expression in rat vascular smooth muscle in
response to in vivo treatment with the organic nitrate ISDN. To induce
tolerance in our model, however, the NTG concentration was 10 µg ·
kg-1 ·
min-1 in rats and 3 to 5 µg ·
kg-1 ·
min-1 in rabbits. NTG concentrations used
in patients range from 0.5 µg · kg-1
· min-1 in patients with
coronary artery disease and myocardial
infarction21 up to 7 µg
· kg-1 ·
min-1 in patients with heart
failure.22 The ISDN
concentrations used in the rat model by Soff et al were 34, 68, and 152
µg · kg-1 ·
min-1. Thus, using much lower NTG
concentrations, we can induce a high degree of tolerance without
alteration of cGK-I expression. With high NTG concentrations, such as
100 µg · kg-1 ·
min-1, however, we found a significant
drop in the expression of cGK-I similar to the extent observed by Soff
et al. This indicates that decreased expression of cGK-I does not
contribute significantly to the development of
tolerance/cross-tolerance in our models but may well occur when very
high concentrations of NO donors are used.
To reconcile our findings with the reduced nitrovasodilator
responsiveness in the setting of NTG tolerance, we assessed the
activity of cGK-I in intact aortic tissue from tolerant and nontolerant
animals. For this purpose, we studied the
phosphorylation of the 46/50-kDa VASP at serine239 to
monitor cGK-I
activity.11 18
VASP is a well-characterized substrate for cGK-I and cAMP-dependent
protein kinase (cAK) in platelets, endothelial
cells, and vascular smooth muscle
cells.12 13 18 23
Functionally, VASP is a crucial factor involved in the regulation of
spatially confined actin
polymerization.18 In
platelets, VASP and VASP phosphorylation are known
to be involved in basal and vasodilator-induced inhibition of integrin
IIbß3 activation and subsequent
aggregation.18 Activation of
cAK and cGK can be analyzed by specific monoclonal antibodies
directed against differently phosphorylated
phospho-VASP forms, because cAK and cGK preferentially
phosphorylate VASP at serine157 and serine239,
respectively.11 18
Experiments with cGK-I–deficient mice established that NO-induced VASP
phosphorylation is mediated primarily by the
cGK-I.12
In the present study, we found significant levels of P-VASP in untreated aortic tissue from rats and rabbits and a striking reduction of P-VASP in aortic tissue from NTG-treated animals compared with untreated controls. The presence of P-VASP in aorta of untreated animals suggests that endogenous cGMP- and/or cAMP-elevating vasodilators (NO and/or prostacyclin) maintain a certain level of P-VASP in the vascular wall. The decrease in P-VASP induced by chronic NTG treatment was not due to decreased availability of this cGK-I substrate, because total VASP expression at the protein level was not different in tolerant and nontolerant aorta. Incubation of control as well as tolerant tissue with the NO donor SNP revealed comparable increases in the phosphorylation of VASP, indicating that the cGMP-signaling pathway downstream of sGC per se is not impaired provided that sufficient NO is available. We have shown previously that cAMP-dependent vasodilation by forskolin is not altered in the setting of nitrate tolerance.4 Furthermore, expression of cAK- and cAMP-mediated VASP phosphorylation in aortic tissue was not significantly affected by NTG pretreatment (data not shown).
These findings clearly indicate that the NO/cGMP pathway is
functionally inhibited in NTG-induced tolerance despite increased
expression of NO-sensitive sGC and normal protein levels of both cGK-I
and VASP. It appears unlikely that activation of a phospho-VASP
phosphatase or an increased activity of a cGK-I inhibitor
or of phosphodiesterases could account for decreased VASP
phosphorylation. This is supported by the observations
that the vascular relaxation in response to direct cGK-I
activators such as
8-bromo-cGMP5 and the extent
of maximal SNP-induced P-VASP
(Figure 5
) are not altered during nitrate
tolerance.
Mechanisms Underlying Inhibition of VASP
Phosphorylation
By using EtBr fluorescence, we were able to
demonstrate (in agreement with previous
observations4 ) that NTG
increases superoxide production not only in
endothelial cells but also in smooth muscle cells. To
identify a possible involvement of a superoxide source, we incubated
intact vascular tissue with 100 µmol/L NADH as described
recently.24 Using this
approach, Guzik et al24
showed that there is a close correlation between NADH-stimulated
superoxide release from intact vessels and from
homogenates, suggesting that use of either approach to
measure NADH oxidase activity seems to be valid. Stimulation of
tolerant but not of control tissue with NADH markedly increased EtBr
fluorescence, compatible with the activation of an NADH-driven
oxidase. Indeed, we recently identified an NADH-dependent
oxidase25 as a superoxide
source in nitrate tolerance as well as decreased activity and/or
expression of the CuZn
SOD.26 In vitro studies
demonstrated a marked regulation of NO-elicited pulmonary
artery relaxation and guanylyl cyclase activation by the NADH oxidase
and SOD.27 Incubation of
vascular homogenates with NADH as well as the inhibition of
SOD by diethyldithiocarbamate strikingly inhibited sGC activity and
NO-mediated vasorelaxation,
respectively.27 Thus, these
findings suggest that an NADH-dependent and SOD-sensitive activity in
the smooth muscle of tolerant aortic tissue inhibits NO-induced sGC
activation to such an extent that cGMP formation is decreased below
nontolerant levels, as observed
experimentally,5 despite a
higher sGC protein expression compared with nontolerant
aorta.
The concept that oxidative stress may, at least in part, be
causally involved in tolerance is further strengthened by the
demonstration that in vitro incubations with vitamin C as well as in
vivo vitamin C treatment partially reversed tolerance, reduced vascular
superoxide, and subsequently improved P-VASP (see
Figure 8).
Conclusions
This study demonstrates an impairment of the NO/cGMP
signaling cascade during nitrate-induced tolerance at the level of
NO-mediated sGC activation. Quantification of P-VASP appears to be a
powerful tool to assess the functional integrity of the NO/cGMP
effector pathway in vascular tissue. The demonstration of increased
superoxide in smooth muscle cells, together with the known
inhibitory effects of superoxide on sGC activity, suggests
that both the oxidative stress concept of tolerance and the
desensitization concept of sGC (as originally proposed by Murad’s
group) may explain, at least in part, the nitrate tolerance
phenomenon.
|
Acknowledgments |
|---|
This study was supported by the Deutsche Forschungsgemeinschaft (Mu 1079/2-2 and SFB 559 and SFB 355) and the German Israel Foundation grant I-504-178 (to Dr Münzel).
Received October 27, 2000; revision received December 1, 2000; accepted December 7, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Feelisch M, Kelm M. Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle cells and endothelial cells. Biochem Biophys Res Commun. 1991;190:286–293.
2. Murad F. Cyclic guanosine monophosphate as a mediator of vasodilation. J Clin Invest. 1986;78:1–5.
3. Lincoln TM, Cornwell TL. Intracellular cyclic GMP receptor proteins. FASEB J. 1993;7:328–338.[Abstract]
4. Munzel T, Sayegh H, Freeman BA, et al. Evidence for enhanced vascular superoxide anion production in nitrate tolerance: a novel mechanism underlying tolerance and cross-tolerance. J Clin Invest. 1995;95:187–194.
5. Molina CR, Andresen JW, Rapoport RM, et al. Effect of in vivo nitroglycerin therapy on endothelium-dependent and independent vascular relaxation and cyclic GMP accumulation in rat aorta. J Cardiovasc Pharmacol. 1987;10:371–378.[Medline] [Order article via Infotrieve]
6. Soff GA, Cornwell TL, Cundiff DL, et al. Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP. J Clin Invest. 1997;100:2580–2587.[Medline] [Order article via Infotrieve]
7. Koesling D, Friebe A. Soluble guanylyl cyclase: structure and regulation. Rev Physiol Biochem Pharmacol. 1999;135:41–65.[Medline] [Order article via Infotrieve]
8. Mülsch A, Busse R, Bassenge E. Desensitization of guanylate cyclase in nitrate tolerance does not impair endothelium-dependent responses. Eur J Pharmacol. 1988;158:191–198.[Medline] [Order article via Infotrieve]
9. Lincoln TM, Pryzwansky KB, Cornwell TL, et al. Cyclic-GMP-dependent protein kinase in smooth muscle and neutrophils. Adv Second Messenger Phosphoprotein Res. 1993;28:121–132.[Medline] [Order article via Infotrieve]
10. Pfeifer A, Klatt P, Massberg S, et al. Defective smooth muscle regulation in cGMP kinase I-deficient mice. EMBO J. 1998;17:3045–3051.[Medline] [Order article via Infotrieve]
11.
Smolenski A,
Bachmann C, Reinhard K, et al. Analysis and regulation of
vasodilator-stimulated phosphoprotein serine 239
phosphorylation in vitro and in intact cells using a
phosphospecific monoclonal antibody.
J Biol Chem. 1998;273:20029–20035.
12.
Massberg S,
Sausbier M, Klatt P, et al. Increased adhesion and aggregation of
platelets lacking cyclic guanosine 3',5'-monophosphate kinase I.
J Exp Med. 1999;189:1255–1264.
13.
Draijer R,
Vaandrager AB, Nolte C, et al. Expression of cGMP-dependent protein
kinase I and phosphorylation of its substrate,
vasodilator-stimulated phosphoprotein, in human
endothelial cells of different origin.
Circ Res. 1995;77:897–905.
14.
Miller FJ Jr,
Gutterman DD, Rios CD, et al. Superoxide production in vascular
smooth muscle contributes to oxidative stress and impaired relaxation
in atherosclerosis. Circ
Res. 1998;82:1298–1305.
15. Munzel T, Li H, Mollnau H, et al. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res. 2000;86:E7–E12.
16. Skatchkov MP, Sperling D, Hink U, et al. Validation of lucigenin as a chemiluminescent probe to monitor vascular superoxide as well as basal vascular nitric oxide production. Biochem Biophys Res Commun. 1999;254:319–324.[Medline] [Order article via Infotrieve]
17.
Kloss S,
Bouloumie A, Mulsch A. Aging and chronic hypertension decrease
expression of rat aortic soluble guanylyl cyclase.
Hypertension. 2000;35:43–47.
18. Reinhard M, Jarchau T, Reinhard K, et al. VASP. In: Kreis T, Vale R, eds. Guidebook to the Cytoskeletal and Motor Proteins. Oxford, UK: Oxford University Press; 1999:168–171.
19. Mülsch A, Bauersachs J, Schäfer A, et al. Effect of YC-1, an NO-independent, superoxide sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators. Br J Pharmacol. 1997;120:681–689.[Medline] [Order article via Infotrieve]
20.
Bauersachs J,
Bouloumie A, Fraccarollo D, et al. Endothelial
dysfunction in chronic myocardial infarction despite increased vascular
endothelial nitric oxide synthase and soluble
guanylate cyclase expression: role of enhanced vascular
superoxide production [see comments].
Circulation. 1999;100:292–298.
21. Jugdutt BI, Warnica JW. Tolerance with low dose intravenous nitroglycerin therapy in acute myocardial infarction. Am J Cardiol. 1989;64:581–587.[Medline] [Order article via Infotrieve]
22. Packer M, Lee W, Kessler PD, et al. Prevention and reversal of nitrate tolerance in patients with congestive heart failure. N Engl J Med. 1987;317:799–804.[Abstract]
23.
Eigenthaler M,
Ullrich H, Geiger J, et al. Defective nitrovasodilator-stimulated
protein phosphorylation and calcium regulation in
cGMP-dependent protein kinase-deficient human platelets of chronic
myelocytic leukemia. J Biol
Chem. 1993;268:13526–13531.
24. Guzik TJ, West NE, Black E, et al. Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors. Circ Res. 2000;86:E85–E90.
25. Munzel T, Kurz S, Rajagopalan S, et al. Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase: a new action for an old drug. J Clin Invest. 1996;98:1465–1470.[Medline] [Order article via Infotrieve]
26. Munzel T, Hink U, Yigit H, et al. Role of superoxide dismutase in in vivo and in vitro nitrate tolerance. Br J Pharmacol. 1999;127:1224–1230.[Medline] [Order article via Infotrieve]
27. Gupte SA, Rupawalla T, Mohazzab HK, et al. Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD. Am J Physiol. 1999;276:H1535–H1542.
This article has been cited by other articles:
 |
|
 |
|
  A. Pautz, P. Rauschkolb, N. Schmidt, J. Art, M. Oelze, P. Wenzel, U. Forstermann, A. Daiber, and H. Kleinert Effects of nitroglycerin or pentaerithrityl tetranitrate treatment on the gene expression in rat hearts: evidence for cardiotoxic and cardioprotective effects Physiol Genomics, July 9, 2009; 38(2): 176 - 185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-S. Tsou, V. Addanki, J. A. Haas, N. A. Page, and H.-L. Fung Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance J. Pharmacol. Exp. Ther., May 1, 2009; 329(2): 649 - 656. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sayed, D. D. Kim, X. Fioramonti, T. Iwahashi, W. N. Duran, and A. Beuve Nitroglycerin-Induced S-nitrosylation and Desensitization of Soluble Guanylyl Cyclase Contribute to Nitrate Tolerance Circ. Res., September 12, 2008; 103(6): 606 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Russo, P. Del Mese, G. Doronzo, L. Mattiello, M. Viretto, A. Bosia, G. Anfossi, and M. Trovati Resistance to the Nitric Oxide/Cyclic Guanosine 5'-Monophosphate/Protein Kinase G Pathway in Vascular Smooth Muscle Cells from the Obese Zucker Rat, a Classical Animal Model of Insulin Resistance: Role of Oxidative Stress Endocrinology, April 1, 2008; 149(4): 1480 - 1489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Hink, A. Daiber, N. Kayhan, J. Trischler, C. Kraatz, M. Oelze, H. Mollnau, P. Wenzel, C. F. Vahl, K. K. Ho, et al. Oxidative Inhibition of the Mitochondrial Aldehyde Dehydrogenase Promotes Nitroglycerin Tolerance in Human Blood Vessels J. Am. Coll. Cardiol., December 4, 2007; 50(23): 2226 - 2232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Wenzel, M. Oelze, M. Coldewey, M. Hortmann, A. Seeling, U. Hink, H. Mollnau, D. Stalleicken, H. Weiner, J. Lehmann, et al. Heme Oxygenase-1: A Novel Key Player in the Development of Tolerance in Response to Organic Nitrates Arterioscler Thromb Vasc Biol, August 1, 2007; 27(8): 1729 - 1735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. J. Perkins, M. Taniguchi, D. O. Warner, E. N. Chini, and K. A. Jones Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L84 - L95. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kollau, M. Beretta, A. C. F. Gorren, M. Russwurm, D. Koesling, K. Schmidt, and B. Mayer Bioactivation of Nitroglycerin by Ascorbate Mol. Pharmacol., July 1, 2007; 72(1): 191 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Esplugues, M. Rocha, C. Nunez, I. Bosca, S. Ibiza, J. R. Herance, A. Ortega, J. M. Serrador, P. D'Ocon, and V. M. Victor Complex I Dysfunction and Tolerance to Nitroglycerin: An Approach Based on Mitochondrial-Targeted Antioxidants Circ. Res., November 10, 2006; 99(10): 1067 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. DiFabio, G. R. Thomas, L. Zucco, M. A. Kuliszewski, B. M. Bennett, M. J. Kutryk, and J. D. Parker Nitroglycerin Attenuates Human Endothelial Progenitor Cell Differentiation, Function, and Survival J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 117 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Schindler, H. Strobel, K. Schonafinger, W. Linz, M. Lohn, P. A. Martorana, H. Rutten, P. W. Schindler, A. E. Busch, M. Sohn, et al. Biochemistry and Pharmacology of Novel Anthranilic Acid Derivatives Activating Heme-Oxidized Soluble Guanylyl Cyclase Mol. Pharmacol., April 1, 2006; 69(4): 1260 - 1268. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Agullo, D. Garcia-Dorado, N. Escalona, M. Ruiz-Meana, M. Mirabet, J. Inserte, and J. Soler-Soler Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes Cardiovasc Res, October 1, 2005; 68(1): 65 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel, A. Daiber, and A. Mulsch Explaining the Phenomenon of Nitrate Tolerance Circ. Res., September 30, 2005; 97(7): 618 - 628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Meurer, S. Pioch, S. Gross, and W. Muller-Esterl Reactive Oxygen Species Induce Tyrosine Phosphorylation of and Src Kinase Recruitment to NO-sensitive Guanylyl Cyclase J. Biol. Chem., September 30, 2005; 280(39): 33149 - 33156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel, A. Daiber, V. Ullrich, and A. Mulsch Vascular Consequences of Endothelial Nitric Oxide Synthase Uncoupling for the Activity and Expression of the Soluble Guanylyl Cyclase and the cGMP-Dependent Protein Kinase Arterioscler Thromb Vasc Biol, August 1, 2005; 25(8): 1551 - 1557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Daiber, M. Oelze, M. Coldewey, M. Bachschmid, P. Wenzel, K. Sydow, M. Wendt, A. L. Kleschyov, D. Stalleicken, V. Ullrich, et al. Oxidative Stress and Mitochondrial Aldehyde Dehydrogenase Activity: A Comparison of Pentaerythritol Tetranitrate with Other Organic Nitrates Mol. Pharmacol., December 1, 2004; 66(6): 1372 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. O. Melichar, D. Behr-Roussel, U. Zabel, L. O. Uttenthal, J. Rodrigo, A. Rupin, T. J. Verbeuren, A. Kumar H. S., and H. H. H. W. Schmidt Reduced cGMP signaling associated with neointimal proliferation and vascular dysfunction in late-stage atherosclerosis PNAS, November 23, 2004; 101(47): 16671 - 16676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Jarvisalo, T. Lehtimaki, and O. T. Raitakari Determinants of Arterial Nitrate-Mediated Dilatation in Children: Role of Oxidized Low-Density Lipoprotein, Endothelial Function, and Carotid Intima-Media Thickness Circulation, June 15, 2004; 109(23): 2885 - 2889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kloss, R. Srivastava, and A. Mulsch Down-Regulation of Soluble Guanylyl Cyclase Expression by Cyclic AMP Is Mediated by mRNA-Stabilizing Protein HuR Mol. Pharmacol., June 1, 2004; 65(6): 1440 - 1451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Gao, S. Dhanakoti, E. M. Trevino, X. Wang, F. C. Sander, A. D. Portugal, and J. U. Raj Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L786 - L792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bertuglia, A. Giusti, and P. Del Soldato Antioxidant activity of nitro derivative of aspirin against ischemia-reperfusion in hamster cheek pouch microcirculation Am J Physiol Gastrointest Liver Physiol, March 1, 2004; 286(3): G437 - G443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Pi, C. Yan, and B. C. Berk Big Mitogen-Activated Protein Kinase (BMK1)/ERK5 Protects Endothelial Cells From Apoptosis Circ. Res., February 20, 2004; 94(3): 362 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Tao, H.-R. Liu, E. Gao, Z.-P. Teng, B. L. Lopez, T. A. Christopher, X.-L. Ma, I. Batinic-Haberle, R. N. Willette, E. H. Ohlstein, et al. Antioxidative, Antinitrative, and Vasculoprotective Effects of a Peroxisome Proliferator-Activated Receptor-{gamma} Agonist in Hypercholesterolemia Circulation, December 2, 2003; 108(22): 2805 - 2811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Hink, M. Oelze, P. Kolb, M. Bachschmid, M.-H. Zou, A. Daiber, H. Mollnau, M. August, S. Baldus, N. Tsilimingas, et al. Role for peroxynitrite in the inhibition of prostacyclin synthase in nitrate tolerance J. Am. Coll. Cardiol., November 19, 2003; 42(10): 1826 - 1834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel, R. Feil, A. Mulsch, S. M. Lohmann, F. Hofmann, and U. Walter Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase Circulation, November 4, 2003; 108(18): 2172 - 2183. [Full Text] [PDF]
|
|
|
|
|
|
A. L. Kleschyov, M. Oelze, A. Daiber, Y. Huang, H. Mollnau, E. Schulz, K. Sydow, B. Fichtlscherer, A. Mulsch, and T. Munzel Does Nitric Oxide Mediate the Vasodilator Activity of Nitroglycerin? Circ. Res., October 31, 2003; 93 (9): e104 - e112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Friebe and D. Koesling Regulation of Nitric Oxide-Sensitive Guanylyl Cyclase Circ. Res., July 25, 2003; 93(2): 96 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Aktas, A. Utz, P. Hoenig-Liedl, U. Walter, and J. Geiger Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets: In Vitro and In Vivo/Ex Vivo Studies Stroke, March 1, 2003; 34(3): 764 - 769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kloss, H. Furneaux, and A. Mulsch Post-transcriptional Regulation of Soluble Guanylyl Cyclase Expression in Rat Aorta J. Biol. Chem., January 17, 2003; 278(4): 2377 - 2383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Gori and J. D. Parker Nitrate Tolerance: A Unifying Hypothesis Circulation, November 5, 2002; 106(19): 2510 - 2513. [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel, I. B. Afanas'ev, A. L. Kleschyov, and D. G. Harrison Detection of Superoxide in Vascular Tissue Arterioscler Thromb Vasc Biol, November 1, 2002; 22(11): 1761 - 1768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Warnholtz, H. Mollnau, T. Heitzer, A. Kontush, T. Moller-Bertram, D. Lavall, A. Giaid, U. Beisiegel, S. L. Marklund, U. Walter, et al. Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits J. Am. Coll. Cardiol., October 2, 2002; 40(7): 1356 - 1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel, A. Mulsch, and A. Kleschyov Mechanisms Underlying Nitroglycerin-Induced Superoxide Production in Platelets: Some Insight, More Questions Circulation, July 9, 2002; 106(2): 170 - 172. [Full Text] [PDF]
|
|
|
|
|
|
H. Mollnau, M. Wendt, K. Szocs, B. Lassegue, E. Schulz, M. Oelze, H. Li, M. Bodenschatz, M. August, A. L. Kleschyov, et al. Effects of Angiotensin II Infusion on the Expression and Function of NAD(P)H Oxidase and Components of Nitric Oxide/cGMP Signaling Circ. Res., March 8, 2002; 90 (4): e58 - e65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Hathaway, D. D. Heistad, D. J. Piegors, and F. J. Miller Jr Regression of Atherosclerosis in Monkeys Reduces Vascular Superoxide Levels Circ. Res., February 22, 2002; 90(3): 277 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Parker and T. Gori Tolerance to the Organic Nitrates: New Ideas, New Mechanisms, Continued Mystery Circulation, November 6, 2001; 104(19): 2263 - 2265. [Full Text] [PDF]
|
|
|
|
|
|
D. Kim, S. D. Rybalkin, X. Pi, Y. Wang, C. Zhang, T. Munzel, J. A. Beavo, B. C. Berk, and C. Yan Upregulation of Phosphodiesterase 1A1 Expression Is Associated With the Development of Nitrate Tolerance Circulation, November 6, 2001; 104(19): 2338 - 2343. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel Does nitroglycerin therapy hit the endothelium? J. Am. Coll. Cardiol., October 1, 2001; 38(4): 1102 - 1105. [Full Text] [PDF]
|
|
|
|
|
|
T. Gori, J. M. Burstein, S. Ahmed, S. E.S. Miner, A. Al-Hesayen, S. Kelly, and J. D. Parker Folic Acid Prevents Nitroglycerin-Induced Nitric Oxide Synthase Dysfunction and Nitrate Tolerance: A Human In Vivo Study Circulation, September 4, 2001; 104(10): 1119 - 1123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Hathaway, D. D. Heistad, D. J. Piegors, and F. J. Miller Jr Regression of Atherosclerosis in Monkeys Reduces Vascular Superoxide Levels Circ. Res., February 22, 2002; 90(3): 277 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Laber, T. Kober, V. Schmitz, A. Schrammel, W. Meyer, B. Mayer, M. Weber, and G. Kojda Effect of Hypercholesterolemia on Expression and Function of Vascular Soluble Guanylyl Cyclase Circulation, February 19, 2002; 105(7): 855 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Schulz, N. Tsilimingas, R. Rinze, B. Reiter, M. Wendt, M. Oelze, S. Woelken-Weckmuller, U. Walter, H. Reichenspurner, T. Meinertz, et al. Functional and Biochemical Analysis of Endothelial (Dys)function and NO/cGMP Signaling in Human Blood Vessels With and Without Nitroglycerin Pretreatment Circulation, March 12, 2002; 105(10): 1170 - 1175. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||