© 1997 American Heart Association, Inc.
Articles |
Nitric Oxide Induces Heme Oxygenase-1 Gene Expression and Carbon Monoxide Production in Vascular Smooth Muscle Cells
From the Houston VA Medical Center (W.D., M.H.K., N.C., K.J.P., A.I.S.) and the Departments of Medicine (W.D., M.H.K., N.C., A.I.S.) and Pharmacology (W.D.), Baylor College of Medicine, Houston, Tex.
Correspondence to Dr William Durante, Houston VA Medical Center, Building 109, Room 116, 2002 Holcombe Blvd, Houston, TX 77030.
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Abstract |
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Abstract Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase–stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-1 (HO-1) mRNA and protein levels in a concentration- and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1ß and tumor necrosis factor-
induced NO
synthesis and also significantly increased the level of HO-1 protein.
The cytokine-stimulated production of both NO and HO-1 protein in
smooth muscle cells was blocked by the NO synthase inhibitor
methyl-L-arginine. These results demonstrate that
exogenously administered or endogenously released NO stimulates HO-1
gene expression and CO production in vascular smooth muscle cells. The
ability of NO to induce HO-catalyzed CO release from vascular smooth
muscle cells provides a novel mechanism by which NO might modulate
soluble guanylate cyclase and, thereby, vascular smooth muscle cell and
platelet function.
Key Words: nitric oxide • carbon monoxide • heme oxygenase
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Introduction |
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Heme oxygenase is the initial and rate-limiting enzyme in heme metabolism, oxidatively degrading heme into biliverdin, iron, and CO.1 At least two distinct isoforms of HO have been identified and cloned.2 3 These isozymes are products of different genes and differ markedly in their tissue expression as well as their molecular properties. The HO-2 isoform is constitutively expressed and is present in high concentration in the brain and testes.4 In contrast, the HO-1 isotype is ubiquitously distributed in mammalian tissues and is strongly and rapidly induced by its own substrate, heme, and by a variety of stress-associated agents, including heavy metals, hyperthermia, hypoxia, and ultraviolet radiation.4 5 Both isoforms of HO are inhibited by various metal protoporphyrins, such as ZnPP or SnPP.4 5
HO plays a critical role in cellular homeostasis by regulating the availability of heme, which serves as the prosthetic moiety of heme proteins. In addition, the capacity of HO to generate CO via heme metabolism may be physiologically important. CO has been identified as an endogenous biological messenger in the brain, and recent studies suggest an important role of CO in hemodynamic regulation.6 Like NO, exogenously administered CO relaxes isolated blood vessels and inhibits platelet aggregation by elevating intracellular levels of cGMP.7 8 9 Moreover, the administration of inducers of HO, such as heme arginate, causes a marked decrease in blood pressure in spontaneously hypertensive rats, whereas HO inhibitors increase blood pressure and peripheral resistance.10 11 In addition, a recent report demonstrated HO activity in vascular endothelium and found that CO release contributes to endothelium-dependent vasodilation.12 These findings indicate that vessel wall–derived CO may serve as an endogenous regulator of vascular tone and platelet reactivity.
In the circulation, NO is a well-established modulator of blood pressure and platelet function.13 Vascular SMCs synthesize NO from the terminal guanidino nitrogen atoms of L-arginine by the iNOS enzyme.14 15 This protein is expressed after exposure of SMCs to various inflammatory cytokines and generates large amounts of NO over prolonged periods of time. More recently, studies in our laboratory and others have demonstrated HO-catalyzed CO production by vascular smooth muscle and associated increases in intracellular cGMP in the same SMCs and in coincubated platelets.16 17 18 Thus, vascular SMCs have the capacity to generate two distinct guanylate cyclase–activating molecules. In the present study, we examined for possible regulatory interactions between these two diatomic signaling gases. We now report that NO, either exogenously administered or endogenously generated from cytokine-treated cells, selectively induces HO-1 gene expression and CO release in vascular SMCs.
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Materials and Methods |
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Materials
FCS, SDS, BSA, DTT, glycerol, trichloroacetic acid, IBMX, N-acetyl-L-cysteine, formamide, polyvinylpyrrolidine, sodium citrate, L-glutamine, L-NMA, penicillin, streptomycin, Tris base, elastase, trypsin, EDTA, collagenase, cycloheximide, dbcGMP, and 8-Br-cGMP were from Sigma Chemical Co; SNP, SIN-1, and SNAP were from Biomol Research Laboratories, Inc; guanidine isothiocyanate, DNase I, sonicated salmon sperm, and CsCl were from GIBCO; actinomycin D, ribonuclease-A, NP-40, PMSF, and ribonuclease T1 were from Boehringer-Mannheim; ZnPP and SnPP were from Porphyrin Products; murine recombinant IL-1ß was from R&D Systems; murine recombinant TNF-
was from Genzyme;
KT5823 was from Calbiochem-Novabiochem Intl; Sephadex G-50 spin columns
were from Bio-Rad Laboratories; T7 RNA polymerase, RNA molecular weight
markers, and antisense GAPDH template were from Ambion Inc; MEM was
from ICN Biomedicals; and [32P]UTP (400 and 3000 Ci/mmol)
was from Amersham.
Cell Culture
SMCs were isolated by elastase and collagenase digestion of rat
thoracic aorta and characterized by morphological and immunological
criteria, as previously described.19 Cells were cultured
serially in MEM containing Earle's salts, 5.6 mmol/L glucose,
2 mmol/L L-glutamine, 20 mmol/L TES-NaOH, 20
mmol/L HEPES-NaOH, 10% (vol/vol) heat-inactivated FCS, 100 U/mL
penicillin, and 100 U/mL streptomycin. Cells were passaged twice a week
by harvesting with trypsin/EDTA and seeded (1:3 ratio) into
75-cm2 flasks. For experiments, subcultured cells were
seeded into multiwell plates or 100-mm culture dishes and used between
passages 6 and 24. When cells reached confluence (
3 or 4 days), the
culture media were replaced with serum-free MEM containing BSA (0.1%
[wt/vol]) for 24 hours and then exposed to the various treatment
regimens.
Protein Analysis
SMCs were scraped in ice-cold PBS, centrifuged at
1000g for 5 minutes at 4°C, and lysed in electrophoresis
buffer (125 mmol/L Tris-HCl [pH 6.8], 12.5% glycerol, 2.5%
DTT, 2% SDS, and trace bromophenol blue). Whole-cell lysates were
boiled for 10 minutes, and SDS-PAGE was performed on 20% gels with 20
µg protein using the buffer system of Laemmli.20 The
separated blots were electrophoretically transferred to nitrocellulose
membranes, as described by Towbin et al.21 Nitrocellulose
blots were blocked for 1 hour in PBS containing 0.1% Tween 20 and 3%
nonfat milk and then incubated with an antibody directed against HO-1
or HO-2 (1:500 dilution) in Tween 20 (0.1%) containing PBS for 1 hour.
The membrane was then washed in PBS and incubated for 1 hour with
anti-rabbit (1:7500 dilution) horseradish peroxidase–conjugated
antibody. After further washing with PBS, blots were incubated in
commercial chemiluminescence reagents (Amersham Corp) and exposed to
photographic film.
Generation of HO Probes
HO cDNA was amplified from RNA derived from rat aortic SMCs by
RT-PCR.22 Primers were designed according to the published
sequence of rat spleen HO-1 and rat testes HO-2.23 24 The
forward 5'-CAGTCGCCTCCAGAGTTTCC-3' and reverse
5'-GTACAAGGAGGCCATCACCAGC-3' primers for HO-1 were used to amplify a
284-bp fragment; the forward 5'-GAGTCGCCTCCAGAGTTTCC-3' and reverse
5'-GAGAGCCAGGCAAGATTCTC-3' primers were used to generate a larger
1070-bp fragment. For HO-2, the forward 5'-CTTACCAAGGAGCAGTCATCTT-3'
and reverse 5'-AAGTCTTTGACAAACTGGGTAT-3' primers were used to amplify a
251-bp fragment. The cDNA (10 µL) was amplified in a 50-µL reaction
volume containing MgCl2 (2.5 mmol/L), a mixture of
dATP, dTTP, dGTP, and dCTP (each at 0.2 mmol/L), HO primers (50 pmol
each), and Taq DNA polymerase (2.5 U/mL) in standard
reaction buffer. Amplification consisted of 30 cycles of PCR (1 minute
at 95°C for denaturing, 1 minute at 58°C for annealing, and 2
minutes and 5 minutes [final cycle] at 72°C for elongation).
Products of PCR amplification were resolved by agarose gel
electrophoresis, stained with ethidium bromide, visualized on an
ultraviolet transilluminator, and photographed. Products of expected
size were subcloned into PCRII plasmids (Invitrogen) and sequenced by
the dideoxy chain-termination method to confirm their identity and
determine their orientation. Antisense RNA probes for HO were generated
in the presence of [32P]UTP (50 µCi) by in vitro
transcription using T7 RNA polymerase.
Ribonuclease Protection Analysis
Total RNA was isolated from SMCs by the guanidine
isothiocyanate/CsCl procedure, and HO mRNA levels were determined by
solution hybridization/ribonuclease protection analysis, as previously
described.25 In brief, total RNA (10 µg) was hybridized
with 1x105 cpm of [32P]UTP-labeled
antisense HO-1 riboprobe and with antisense GAPDH (316-bp) RNA to
control for variations in the amount of RNA used in each assay. Samples
were incubated in hybridization buffer (65 mmol/L sodium citrate,
200 mmol/L sodium acetate, 0.5 mmol/L EDTA, and 55%
formamide) for 16 hours at 45°C, followed by digestion with
ribonuclease A (4 µg/mL) and ribonuclease 1 (0.2 µg/mL) at room
temperature for 30 minutes. Protected RNA was analyzed by
electrophoresis using 6% acrylamide/8 mol per L urea gel. The gel was
exposed overnight to x-ray film at -70°C in the presence of
intensifying screens. The size of the predicted nucleotide-protected
fragments was confirmed by using a 32P-labeled RNA
ladder.
Nuclear Run-on Transcription Assay
Vascular SMCs were harvested in ice-cold PBS, centrifuged at
1000g for 5 minutes at 4°C, lysed in NP-40 buffer (10
mmol/L Tris-HCl [pH 7.4], 10 mmol/L NaCl, 3 mmol/L
MgCl2, 0.5% NP-40, 1 mmol/L PMSF, and 1 mmol/L
DTT), and left on ice for 5 minutes. The cells were homogenized, and
the nuclei were sedimented by centrifugation (1000g for 5
minutes at 4°C), suspended in storage buffer (40% glycerol, 50
mmol/L Tris-HCl [pH 8.3], 5 mmol/L MgCl2, and
0.1 mmol/L EDTA), and stored at -70°C until used.
Nuclei (50 µL) were suspended, in a final volume of 200 µL, with
transcription buffer (25 mmol/L Tris-HCl [pH 7.5], 140
mmol/L KCl, 2.5 mmol/L MgCl2, 0.5 mmol/L
MnCl2, 0.1 mmol/L DTT, 10% glycerol, 1 mmol/L
each of ATP, GTP, and CTP, and 250 µCi of [-32P]UTP)
and incubated at 30°C for 30 minutes. After DNase I treatment (200
U/mL), the mixture was deproteinized with proteinase K (1.2 µg), and
the elongated transcripts were purified by phenol-chloroform
extraction, spin-column chromatography (Sephadex G-50), and ethanol
precipitation. Labeled RNA (5x106 cpm/mL) was hybridized
to 5 µg each of linearized denatured cDNA plasmids, which were
immobilized on Genescreen Plus membranes (New England Nuclear-Dupont).
The plasmid cDNAs used were the rat HO-1 and GAPDH, and the plasmid
PCRII without any insert was used as the negative control. The
membranes were incubated at 42°C for 48 hours in hybridization buffer
(40% formamide, 0.2% polyvinylpyrrolidine, 2x SSC [1.5 mol/L NaCl
and 0.15 mol/L sodium citrate, pH 7.0], 1% SDS, and 100 µg/mL
salmon sperm). After hybridization, membranes were extensively washed
(0.1x SSC and 0.1% SDS) at 55°C and exposed to x-ray film at
-70°C in the presence of intensifying screens. After
autoradiography, each dot was carefully excised, and radioactivity was
determined by scintillation counting. For HO-1, specific bound counts
were determined by subtracting counts bound to the control plasmid
PCRII and then normalized with respect to the GAPDH signal.
CO Detection System
CO release by vascular SMCs was determined using a previously
described bioassay consisting of a coincubation system in which cGMP
production in "detector" platelets layered in suspension over
monolayers of SMCs reflects the activation of platelet-soluble
guanylate cyclase by CO released from the SMCs.16
Platelets were isolated from venous blood obtained from healthy
drug-free donors, and washed platelet suspensions in Tyrode's buffer
(mmol/L: NaCl 130, Tris base 10, NaHCO3 9, glucose 6, KCl
3, CaCl2 1, MgCl2 0.9, and
KH2PO4 0.8; pH 7.4) were prepared as previously
described.26 The washed platelet suspensions were then
treated with IBMX (0.1 mmol/L) and added onto monolayers of SMCs.
In some experiments, hemoglobin (50 µmol/L) was added to the
platelet suspension. Before the addition of the platelet suspension to
the SMCs, the medium was aspirated, and the cells were thoroughly
washed with PBS to ensure that platelets were not exposed to any of the
treatment compounds. After 45 minutes of coincubation with SMCs, the
platelet suspensions were collected in trichloroacetic acid (6%
[wt/vol]), briefly sonicated, and pelleted in a microfuge. Platelet
lysates were then extracted with 4 vol of water-saturated ether and
assayed for cGMP using a commercially available radioimmunoassay kit
(New England Nuclear-Dupont).
Nitrite
The generation of NO was determined by measuring the release of
nitrite, the stable oxidation product of NO.27 Sample
aliquots (0.4 mL) were mixed with an equal volume of Greiss reagent
(1% sulfanilamide and 1% naphthylethylenediamine dihydrochloride in
2% phosphoric acid), the mixture was incubated at room temperature for
10 minutes, and the absorbance was measured at 540 nm.28
Nitrite concentrations were determined relative to a standard curve by
using an aqueous solution of sodium nitrite, and background nitrite
values corresponding to serum-free medium were subtracted from
experimental values.
Statistics
Results are expressed as mean±SEM. Statistical analysis was
performed with the use of a Student's two-tailed t test and
an ANOVA when more than two treatments were compared. Values of
P<.05 were considered statistically significant.
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Results |
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Treatment of rat aortic SMCs with an NO donor, SNP (1 mmol/L), SNAP (1 mmol/L), or SIN-1 (1 mmol/L), stimulated the expression of HO-1 mRNA without affecting the steady state level of HO-2 message (Fig 1A
). Although HO-1 mRNA appears absent
in untreated control cells, a faint transcript is apparent after
prolonged exposure of the autoradiogram (data not shown).
Immunoblotting with HO-1 or HO-2 antibodies identified single bands
with approximate molecular masses of 30 and 36 kD, which correspond to
the estimated sizes of HO-1 and HO-2, respectively (Fig 1B).16 Incubation of SMCs with the NO donors selectively
stimulated the production of HO-1 protein (Fig 1B). The capacity of SNP
(0.1 to 1.0 mmol/L) to increase HO-1 mRNA and protein levels in
SMCs was both concentration and time dependent (Figs 2 and 3). SNP (1 mmol/L) elevated HO-1 mRNA within 2
hours, became maximal at 4 hours, and gradually declined over 24 hours.
In contrast, HO-1 protein levels increased 4 hours after SNP treatment,
and the level progressively increased over 24 hours (Fig 3). In some
instances, SNP also induced the expression of a 27-kD protein, which
likely represents a previously described proteolytic breakdown product
of HO-1.29 Similar results were also obtained with SNAP or
SIN-1 (data not shown).
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In the next series of experiments, the molecular mechanism by which NO
stimulates HO-1 expression was determined. Treatment of SMCs with
cycloheximide (5 µg/mL) and actinomycin D (2 µg/mL) blocked
SNP-stimulated HO-1 mRNA and protein expression (Fig 4).
Subsequently, nuclear run-on studies were conducted with nuclei
isolated from SMCs that had been treated with NO donors (1 mmol/L)
for 4 hours. Fig 5 demonstrates that SNP (1
mmol/L), SNAP (1 mmol/L), or SIN-1 (1 mmol/L) increased HO-1
gene transcription between 3- and 6-fold. In contrast, NO donors had no
effect on the stability of HO-1 mRNA. Incubation of SMCs with
actinomycin D (2 µg/mL) resulted in a decay of HO-1 message with a
half-life of 3 hours, and this remained unchanged in the presence of
the various NO donors (data not shown). Treatment of SMCs with a
membrane-permeable cGMP analogue, dbcGMP (1 mmol/L) or 8-Br-cGMP
(1 mmol/L), did not stimulate HO-1 expression (Fig 6). Furthermore, the phosphodiesterase inhibitor IBMX
(0.1 mmol/L) did not affect NO-mediated HO-1 expression (data not
shown). Similarly, the highly selective cGMP-dependent protein kinase
inhibitor KT5823 (20 µmol/L) failed to inhibit NO-induced HO-1
gene expression (data not shown). Finally, treatment of SMCs with
N-acetyl-L-cysteine (10 mmol/L) had no
effect on NO-stimulated HO-1 expression (data not shown).
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In subsequent experiments, HO activity was measured by monitoring SMC
CO production. Since CO is a readily diffusible membrane-soluble gas
that is known to activate soluble guanylate cyclase,8 9 16
HO activity was determined by measuring the intracellular concentration
of cGMP in coincubated detector platelets. Incubating platelets with
SMCs that had been treated with SNP (1 mmol/L), SNAP-1 (1
mmol/L), or SIN-1 (1 mmol/L) for 24 hours resulted in an 4-fold
greater increase in platelet cGMP concentration than that found in
platelets exposed to untreated control cells (Fig 7A).
The stimulatory effect on platelet cGMP concentration by SNP-treated
(1 mmol/L) SMCs was blocked by incubating the vascular cells with
an HO inhibitor, ZnPP (20 µmol/L) or SnPP (20 µmol/L), or
by adding the CO scavenger, hemoglobin (50 µmol/L), to platelets
during their incubation with SNP-treated SMCs (Fig 7B). In contrast,
treatment of SMCs with L-NMA (1 mmol/L) did not alter platelet
cGMP levels under any condition (data not shown). In the absence of NO
treatment, exposure of SMCs to the metalloprotoporphyrins resulted in a
minor decrease in intracellular cGMP concentration of incubated
platelets (data not shown).
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Finally, incubating SMCs with a cytokine mixture of IL-1ß (10 ng/mL)
and TNF- (10 ng/mL) for 24 hours induced nitrite generation and also
significantly increased HO-1 protein (Fig 8). The
addition of L-NMA (1 mmol/L) to SMCs abolished the
cytokine-induced production of nitrite and markedly reduced the
cytokine-stimulated rise in HO-1 protein (Fig 8). In the absence of
cytokines, L-NMA had no effect on SMC nitrite production or HO-1
protein levels (Fig 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates that NO selectively induces the expression of the HO-1 gene and the production of CO in vascular SMCs. Treatment of vascular SMCs with three structurally dissimilar NO donors stimulates HO-1 mRNA and protein production in a concentration- and time-dependent manner, with the elevation in HO-1 message preceding the increase in HO-1 protein. NO-induced HO-1 gene expression is dependent on de novo RNA and protein synthesis and arises via the transcriptional activation of the gene without any accompanying changes in the stability of HO-1 mRNA.
The NO-induced increase in HO-1 gene expression is associated with an increase in HO activity as measured by CO production. Incubating platelets with NO-treated SMCs results in a significantly greater increase in platelet cGMP concentration than that found in platelets exposed to untreated control SMCs. The SMC-induced rise in platelet cGMP results from increased HO activity, since the HO inhibitors, ZnPP and SnPP, abrogate the cGMP-elevating effect of NO-treated cells. In addition, the CO and NO scavenger, hemoglobin, reverses the increase in platelet cGMP in platelets exposed to NO-treated SMCs, whereas the NO synthase inhibitor L-NMA was without effect. These results demonstrate that SMC-derived CO is responsible for the elevation in platelet cGMP concentration. The ability of NO to stimulate heme oxygenase activity in vascular SMCs complements a recent study demonstrating NO-mediated activation of heme oxygenase in vascular endothelium.30 The capacity of NO to induce CO release from vascular cells may provide an additional mechanism by which NO activates soluble guanylate cyclase and regulates vascular tone. In addition, vessel wall–derived CO may also regulate platelet reactivity. Although the high levels of hemoglobin in circulating erythrocytes would scavenge CO released into the circulation, erythrocytes tend to stream to the center of the lumen, whereas platelets are distributed primarily near the vessel wall.31 32 Thus, platelets localized to sites of vessel wall injury may be exposed to an inhibitory concentration of CO.
The physiological relevance of NO-induced HO-1 gene expression was
further underscored in the present study by the capacity of
endogenously released NO to stimulate HO-1 expression. Treatment of
SMCs with the inflammatory cytokines IL-1ß and TNF- stimulates NO
synthesis and significantly increases HO-1 protein levels. The addition
of L-NMA to SMCs completely blocks the cytokine-induced production of
NO and markedly attenuates the cytokine-stimulated rise in HO-1
protein, indicating that SMC NO synthesis stimulates HO-1 expression.
Interestingly, L-NMA fails to completely reverse the effect of the
cytokines, suggesting that there is also a minor NO-insensitive pathway
by which cytokines can induce HO-1 expression in these cells.
Alternatively, it is possible that the blockade of iNOS by L-NMA is not
complete and that small amounts of NO are being generated by SMCs that
are not being measured by the nitrite assay.
The mechanism by which NO induces HO-1 gene expression is not entirely clear. It does not involve the cGMP signaling pathway, since lipophilic analogues of cGMP fail to stimulate HO-1 expression and inhibitors of cGMP breakdown or cGMP kinase do not affect NO-mediated HO-1 gene induction. Although oxidative stress is a well-established inducer of HO-1 expression,33 it does not appear that this mediates the NO effect, since the antioxidant N-acetyl-L-cysteine fails to regulate NO stimulated HO-1 gene expression. Interestingly, a recent study demonstrated that NO causes the release of free heme from heme proteins.34 Since free heme is known to transcriptionally upregulate its own degradation by HO, the liberation of heme by NO may induce HO-1 expression.4 5
NO has been shown to have markedly different biological effects depending on its redox state.35 In the present study, three different compounds were used that generate alternative redox forms of NO. Whereas SNAP releases the free radical NO, SIN-1 generates both NO and superoxide, which can subsequently combine to form peroxynitrite.36 37 In contrast, SNP generates the nitrosonium ion (NO+).38 Since all three NO donors are capable of stimulating HO-1 expression, it appears that induction of HO-1 gene expression is independent of the redox state. However, these exogenously applied NO congeners readily undergo interconversion from one redox form to another, depending on the ambient redox milieu.35 Similarly, iNOS produces different redox species of NO depending on the intracellular redox conditions.39 This complex redox chemistry of NO makes it difficult to precisely determine which redox form(s) is involved in regulating HO-1 gene expression.
The induction of HO activity by NO in vascular SMCs may be of pathophysiological significance. In response to vascular injury, SMCs express iNOS,40 41 a high output isoform of NOS that is capable of generating cytotoxic amounts of NO.14 15 The release of CO following HO activation may provide an important mechanism in limiting NO release, since CO directly inhibits iNOS activity by binding to the heme moiety of the enzyme.42 In addition, because heme is essential for both the intracellular assembly of the active dimeric iNOS and its catalytic activity,43 increases in HO activity may reduce intracellular heme levels, thereby limiting the formation of active iNOS enzyme. In this regard, significantly lower cellular heme levels are found after iNOS induction in rat hepatocytes and macrophages.34 44 Thus, NO-induced HO-1 gene expression may provide an important negative-feedback regulatory mechanism limiting the release of cytotoxic levels of NO by SMCs while still preserving blood flow at sites of vascular injury by releasing guanylate cyclase–stimulatory CO.
Finally, NO-induced HO activity may also provide vascular SMCs with cytoprotection from the oxidative stress associated with elevated rates of NO synthesis.45 46 Induction of HO activity results in the generation of biliverdin, which is subsequently converted to bilirubin by biliverdin reductase.1 Both these heme metabolites are efficient scavengers of reactive oxygen species and inhibit lipid peroxidation.47 48 Interestingly, a recent study has correlated elevations in serum bilirubin concentration with a significant and marked reduction in the risk of coronary artery disease.49 Thus, NO-stimulated HO activity and the subsequent formation of bilirubin by vascular SMCs may provide blood vessels an important cellular defense mechanism against oxidative tissue injury.
In conclusion, the present study demonstrates that NO induces the transcriptional activation of the HO-1 gene and the generation of CO in vascular SMCs. Induction of HO activity may play an important cytoprotective role by limiting iNOS activation and by synthesizing antioxidant molecules. In addition, the HO-catalyzed production of guanylate cyclase–stimulatory CO may provide vascular SMCs an additional mechanism by which blood flow and fluidity are maintained at sites of vascular injury.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by National Heart, Lung, and Blood Institute grant HL-36045, the Veterans Affairs Merit Review Board, and a Grant-in-Aid from the American Heart Association, Texas Affiliate, Inc. Dr Kroll is an Established Investigator of the American Heart Association. The authors thank Dr Roger K. Sunahara for designing the various HO primers and Lan Liao for expert technical assistance.
Received September 16, 1996; accepted January 15, 1997.
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S. D. Idriss, T. Gudi, D. E. Casteel, V. G. Kharitonov, R. B. Pilz, and G. R. Boss Nitric Oxide Regulation of Gene Transcription via Soluble Guanylate Cyclase and Type I cGMP-dependent Protein Kinase J. Biol. Chem., April 2, 1999; 274(14): 9489 - 9493. [Abstract] [Full Text] [PDF]
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R. C.M. Siow, H. Sato, and G. E. Mann Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide? Cardiovasc Res, February 1, 1999; 41(2): 385 - 394. [Abstract] [Full Text] [PDF]
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M. J. J. Vesely, D. J. Exon, J. E. Clark, R. Foresti, C. J. Green, and R. Motterlini Heme oxygenase-1 induction in skeletal muscle cells: hemin and sodium nitroprusside are regulators in vitro Am J Physiol Cell Physiol, October 1, 1998; 275(4): C1087 - C1094. [Abstract] [Full Text] [PDF]
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R. Motterlini, A. Gonzales, R. Foresti, J. E. Clark, C. J. Green, and R. M. Winslow Heme Oxygenase-1–Derived Carbon Monoxide Contributes to the Suppression of Acute Hypertensive Responses In Vivo Circ. Res., September 7, 1998; 83(5): 568 - 577. [Abstract] [Full Text] [PDF]
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I. J. Benjamin and D. R. McMillan Stress (Heat Shock) Proteins : Molecular Chaperones in Cardiovascular Biology and Disease Circ. Res., July 27, 1998; 83(2): 117 - 132. [Abstract] [Full Text] [PDF]
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C. Bouton and B. Demple Nitric Oxide-inducible Expression of Heme Oxygenase-1 in Human Cells. TRANSLATION-INDEPENDENT STABILIZATION OF THE mRNA AND EVIDENCE FOR DIRECT ACTION OF NITRIC OXIDE J. Biol. Chem., October 13, 2000; 275(42): 32688 - 32693. [Abstract] [Full Text] [PDF]
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N. Hill-Kapturczak, L. Truong, V. Thamilselvan, G. A. Visner, H. S. Nick, and A. Agarwal Smad7-dependent Regulation of Heme Oxygenase-1 by Transforming Growth Factor-beta in Human Renal Epithelial Cells J. Biol. Chem., December 22, 2000; 275(52): 40904 - 40909. [Abstract] [Full Text] [PDF]
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S. Uma, B.-G. Yun, and R. L. Matts The Heme-regulated Eukaryotic Initiation Factor 2alpha Kinase. A POTENTIAL REGULATORY TARGET FOR CONTROL OF PROTEIN SYNTHESIS BY DIFFUSIBLE GASES J. Biol. Chem., April 27, 2001; 276(18): 14875 - 14883. [Abstract] [Full Text] [PDF]
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