(Received for publication, November 15, 1996, and in revised form, December 3, 1996)
From the Cardiovascular Biology Laboratory, Endotoxic shock is a life-threatening consequence
of severe Gram-negative infection characterized by vascular smooth
muscle cell relaxation and severe hypotension. The production of nitric oxide (NO), through the inducible NO synthase pathway, has been implicated as a major contributor in this process. We now demonstrate that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO)
in the course of heme metabolism, may also be involved in the
hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) mRNA
levels are dramatically increased in aortic tissue from rats receiving
endotoxin, and this increase in vascular HO-1 message is associated
with an 8.9-fold increase in HO enzyme activity in vivo.
Immunocytochemical staining localizes an increase in HO-1 protein
within smooth muscle cells of both large (aorta) and small (arterioles)
blood vessels. Furthermore, zinc protoporphyrin IX, an inhibitor of HO
activity, abrogates endotoxin-induced hypotension in rats. Studies
performed in rat vascular smooth muscle cells in vitro show
that the induction of HO-1 mRNA is regulated at the level of gene
transcription, and this induction is independent of NO production.
Taken together, these studies suggest that the up-regulation of HO-1,
and the subsequent production of CO, contributes to the reduction in
vascular tone during endotoxic shock.
Endotoxemia leading to shock is a detrimental consequence of
severe Gram-negative bacterial infection. Endotoxic shock is initiated
by the release of bacterial cell wall-derived lipopolysaccharide (LPS)1 and the subsequent production of
cytokines and vasoactive mediators that result in vascular smooth
muscle cell relaxation and hypotension (1, 2). One of the most
important cytokines in the cascade of events leading to LPS-induced
hypotension is interleukin (IL)-1 HO is the enzyme that generates carbon monoxide (CO) and biliverdin
(subsequently reduced to bilirubin) in the course of heme metabolism
(9). CO is a gas molecule that shares some of the properties of NO,
inasmuch as CO binds to the heme moiety of cytosolic guanylyl cyclase
to produce cGMP (10). Two distinct forms of heme oxygenase have been
identified (9): HO-1 (an inducible isozyme) and HO-2 (a non-inducible
isozyme). Morita and colleagues (11) have demonstrated that HO-1 is
induced by hypoxia in vascular smooth muscle cells in vitro,
and that its product, CO, promotes the accumulation of cGMP in this
cell type. The investigators also showed that smooth muscle
cell-derived CO inhibits the production of endothelium-derived
vasoactive agents (such as endothelin-1 and platelet-derived growth
factor-B) under hypoxic conditions (12). Furthermore, a recent study by
Raju and Maines (13) demonstrated that expression of HO-1 in the
cardiovascular system is up-regulated in vivo using a model
of acute renal ischemia and reperfusion. The authors speculated that
increased HO-1 expression, and the ensuing CO production, may promote
vasodilation as a defense response to renal ischemia/reperfusion. These
previous studies (both in vitro and in vivo)
suggest a physiologic role for CO in vascular biology.
We designed the present study to further understand the role of CO in
endotoxic shock by 1) analyzing the regulation of vascular HO-1
in vivo, 2) administering zinc protoporphyrin IX (ZnPP), an
inhibitor of HO activity, to rats made hypotensive by LPS, and 3)
investigating the mechanism of HO-1 induction in vascular smooth muscle
cells in vitro.
Salmonella typhosa LPS
(Sigma) was dissolved in 0.9% saline and stored at
Rat aortic smooth muscle cells (RASMC) were
harvested from male Sprague-Dawley rats (Charles River Laboratories,
Wilmington, MA) by enzymatic dissociation according to the method of
Gunther et al. (16). The cells were cultured in Dulbecco's
modified Eagle's medium (JRH Biosciences, Lenexa, KS) and supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and 25 mM Hepes (pH 7.4)
(Sigma). RASMC were passaged every 4-7 days, and
experiments were performed on cells 6-8 passages from primary culture.
After the cells had grown to confluence, they were placed in 2% fetal
calf serum 12 h before the experiments.
Total RNA was obtained from rat
aortas and cultured smooth muscle cells by guanidinium isothiocyanate
extraction and centrifuged through cesium chloride (17). The RNA was
fractionated on a 1.3% formaldehyde-agarose gel and transferred to
nitrocellulose filters. The filters were hybridized at 68 °C for
2 h with 32P-labeled rat HO-1 or HO-2 probes (11) in
QuikHyb solution (Stratagene, La Jolla, CA). The hybridized filters
were then washed in 30 mM sodium chloride, 3 mM
sodium citrate, and 0.1% sodium dodecyl sulfate solution at 55 °C
and autoradiographed with Kodak XAR film at RASMC were either not stimulated
(vehicle) or stimulated with IL-1 Aortas were harvested from male
Sprague-Dawley rats (200-250 g) treated with vehicle or LPS (4 mg/kg
intravenously), the adventitia was stripped, and the tissue was
homogenized with PolytronTM in homogenization buffer (30 mM
Tris, pH 7.5, 0.25 M sucrose, 0.15 M NaCl)
containing CompleteTM protease inhibitor (Boehringer Mannheim). The
homogenate was centrifuged at 10,000 × g for 15 min,
and the supernatant fraction was subsequently centrifuged at
100,000 × g for 1 h. The microsomal pellet was
resuspended in 50 mM potassium phosphate buffer (pH 7.4)
containing CompleteTM protease inhibitor. HO enzyme activity was
measured by bilirubin generation as described (19, 20). The liver
microsomal supernatant fraction from the control animal served as the
source of biliverdin reductase. A reaction mixture (0.5 ml) containing
33 µM hemin, rat liver microsomal supernatant fraction
(0.3 mg), NADPH generating system, and aortic microsomal protein was
incubated at 37 °C for 10 min in the dark. The reaction mixture
without NADPH generating system served as a blank. The reactions were
stopped by placement on ice, and subsequently scanned with a
spectrophotometer (Beckman, Columbia, MD). The amount of bilirubin
formed was determined as the difference in optical density units
between 462 and 530 nm (extinction coefficient, 40 nm Male Sprague-Dawley rats
(200-250 g) treated with LPS or vehicle were perfused with 4%
paraformaldehyde. The aortas were removed, post-fixed with 4%
paraformaldehyde overnight at 4 °C, and then soaked in 30% sucrose
for 2 days at 4 °C. The specimens were cut at a thickness of 5 µm.
Immunocytochemical procedure was performed as described (21, 22). To
reduce nonspecific binding, the sections were incubated in
phosphate-buffered saline containing 10% normal goat serum and 0.4%
Triton X-100 for 30 min. Rabbit polyclonal antibody against purified
rat liver HO-1 (StressGen Biotechnologies, Victoria, BC, Canada) was
applied for 1 h at room temperature and then overnight at 4 °C
at a dilution of 1:1000-1200. Sections were rinsed twice with high
salt phosphate-buffered saline (0.5 M NaCl) and once with
regular phosphate-buffered saline (5 min for each wash) and then
incubated with biotinylated goat anti-rabbit IgG at a dilution of 1:500
for 1 h at room temperature. They were then rinsed with phosphate
buffered saline and incubated with avidin-biotin complex (ABC elite
kit, Vector Laboratories, Burlingame, CA) at a dilution of 1:100 for
1 h at room temperature. After washing with PBS, the tissue
sections were treated with diaminobenzidine in phosphate buffered
saline-H2O2 for 1-3 min using the peroxidase substrate kit DAB (Vector) and then transferred into phosphate buffered
saline solution to stop the reaction. The presence of HO-1 was
indicated by the development of a brown color within the cytoplasm.
Counterstaining was performed with 0.5% methyl green.
To determine the amount of NO produced by
RASMC, we measured a stable product of NO oxidation,
NO2 Male Sprague-Dawley rats (Charles
River Laboratories) weighing 175-200 g received an intraperitoneal
injection of thiobutabarbital sodium (100 mg/kg), which kept them
anesthetized throughout the experiment. The trachea was cannulated with
a tubing adapter. Then the right carotid artery was cannulated with
PE-50 tubing to measure arterial pressure (24, 25) using MacLab
monitoring equipment from ADInstruments, Inc. (Milford, MA). Saline
(0.9%) was used as a control for LPS and ZnPP. The LPS group received LPS (4 mg/kg intra-arterially) and saline vehicle in place of ZnPP. The
LPS+ZnPP groups received LPS (4 mg/kg intra-arterially) followed by
ZnPP (10 µmol/kg or 1 µmol/kg intraperitoneally). The ZnPP group
received ZnPP (10 µmol/kg intraperitoneally) and saline vehicle in
place of LPS. After an initial 25% decrease in mean arterial pressure
(corresponding to time point 0) in the rats receiving LPS (LPS group
and LPS+ZnPP groups), ZnPP (or saline vehicle) was administered and
mean arterial pressure was monitored over the next 90 min. Preliminary
experiments revealed that all rats responded to LPS with time; however,
if a rat's mean arterial pressure did not decrease within 2 h, it
was excluded from the study. Thus, only the rats most sensitive to LPS
were used and it was not necessary to prolong their time under
anesthesia.
Comparisons between the vehicle and
LPS-treated groups for HO enzyme activity were made using unpaired
t tests (two-tailed). Comparisons of hemodynamic data
between groups were made by factorial analysis of variance followed by
Fisher's least significant difference test, or unpaired t
tests. Statistical significance was accepted for a p
value < 0.05.
To determine if LPS regulates vascular HO-1 in an animal
model of endotoxic shock, we injected rats with vehicle or S. typhosa LPS (4 mg/kg intravenously). HO-1 mRNA levels were
markedly increased in aortic tissue after 9 h of LPS stimulation
compared with tissue from rats receiving vehicle (Fig.
1A). We have demonstrated previously that
this dose of LPS produces hypotension in rats (25), and the 9-h time
point was chosen after performing an in vivo time-course experiment to assess maximal HO-1 mRNA induction. Moreover, LPS induced HO-1 message as early as 2 h after stimulation, and LPS did not increase HO-2 mRNA levels (data not shown).
We next assessed whether the increase in HO-1 mRNA levels
corresponded to an increase in HO enzyme activity. Rats were given vehicle or LPS (4 mg/kg intravenously), and the aortas were harvested 9 h later (adventitia of the vessels was stripped prior to
analysis). LPS promoted an 8.9-fold increase (p < 0.05) in HO enzyme activity (Fig. 1B). In fact, the level of
HO enzyme activity in the aortic tissue from rats receiving LPS
(36.3 ± 2.4 nmol/mg of protein/h) was comparable to the level of
HO activity in the liver of control rats (40 ± 2.1 nmol/mg of
protein/h, p = 0.34). The activity of HO is typically
highest in organs rich in reticuloendothelial cells (i.e.
liver, spleen, and bone marrow) (9, 10). These data demonstrate the
significant amount of inducible HO enzyme activity generated in
vascular tissue after LPS stimulation. Preliminary experiments were
also performed to determine a dose of ZnPP that would suppress, but not
abolish, vascular HO activity in vivo. When ZnPP was
administered at a dose of 10 µmol/kg (intraperitoneally) 1 h
after LPS, the level of vascular HO enzyme activity (6.1 nmol/mg of
protein/h) was very similar to that of control rats (Fig.
1B). Thus, ZnPP doses of 10 and 1 µmol/kg were used in
subsequent hemodynamic experiments.
To localize the cell type within the vessel responsible for the
increase in HO enzyme activity, immunocytochemical staining was
performed using a rabbit anti-HO-1 antibody. Staining for HO-1 protein
was increased in the smooth muscle cells of aortas from rats receiving
LPS (Fig. 2B) compared with rats receiving vehicle (Fig. 2A). Moreover, immunocytochemical staining
demonstrated an LPS-induced increase in HO-1 expression in the smooth
muscle cells of arterioles (Fig. 2, E and F
compared with C and D), smaller vessels that
contribute to the regulation of vascular tone. Staining for HO-1 was
also increased in the endothelium of aortas and arterioles after LPS
stimulation. These data demonstrate an increase in vascular HO-1
mRNA and protein levels after LPS administration in vivo and, more importantly, an increase in HO enzyme activity.
To provide
evidence that HO contributes to the reduction in arterial pressure
associated with endotoxemia, we gave ZnPP to rats made hypotensive by
LPS. S. typhosa LPS given at a dose of 4 mg/kg
intra-arterially produced profound and reproducible hypotension in male
Sprague-Dawley rats (LPS group, Fig. 3). We then gave the same dose of LPS to another group of rats, followed by ZnPP (10 or
1 µmol/kg intraperitoneally) after an initial 25% decrease in mean
arterial pressure (LPS+ZnPP groups, Fig. 3). By the time the arterial
pressure had decreased by 25%, HO-1 mRNA levels had increased by
4-fold (Fig. 3, inset). ZnPP given at a dose of 10 µmol/kg
after the onset of LPS-induced shock abolished the hypotension and
produced a significantly higher mean arterial pressure than the LPS
group after 30, 60, and 90 min (p < 0.05). ZnPP
administered at a dose of 1 µmol/kg arrested the LPS-induced
hypotension, but the response was significantly less than in the higher
dose (10 µmol/kg) LPS+ZnPP group after 30 and 60 min
(p < 0.05). ZnPP did not significantly increase mean
arterial pressure when given at the dose of 10 µmol/kg
intraperitoneally to rats not receiving LPS (ZnPP group, Fig. 3). The
lack of an increase in arterial pressure again suggests this dose of
ZnPP (10 µmol/kg) did not completely inhibit HO enzyme activity. Mean
arterial pressure was not altered in control rats receiving vehicles in
the place of LPS and ZnPP (data not shown).
To further understand the molecular
mechanisms regulating vascular HO-1, we investigated the induction of
HO-1 mRNA by IL-1
To
determine the mechanism by which IL-1
Investigators have recently suggested that NO
itself can induce HO-1 in vitro (26). Thus, to determine if
an increase in NO production contributed to the induction of HO-1
mRNA, RASMC were treated with IL-1
CO, a gas formed endogenously from heme metabolism, shares many of
the chemical and biological properties of NO (10). The physiologic
importance of NO and the enzymes that foster the production of NO (NOS)
have been studied in detail (5-8, 28); however, much less is known
about the physiologic function of CO and the heme degradative enzymes
HO-1 and HO-2. A recent study (29) showed prominent immunocytochemical
staining for HO-2 in the vascular endothelium under basal conditions.
HO-2 staining was also present in adventitial nerves of blood vessels
and in neurons in the autonomic ganglia. The location of the staining
for HO-2 was comparable with constitutive isoforms of NOS, and
inhibitors of HO enzyme activity were able to partially attenuate
endothelium-dependent vasodilation. This study implied
complementary and possibly related roles for HO-2 and constitutive
isoforms of NOS (29). Studies have also demonstrated that CO can be
produced by arteries in vivo (30, 31), and higher doses of
HO inhibitors (such as ZnPP, 45 µmol/kg) induce an increase in
arterial pressure in rats (32). Taken together these studies suggest
CO, generated by HO-2, may contribute to the regulation of vascular
tone under basal conditions. However, the role of the inducible isoform
of HO, HO-1, in vascular biology and pathophysiology is less clear.
To gain more insight into the potential role of CO in the regulation of
vascular tone during endotoxic shock (a cytokine-driven disease
process; Refs. 1 and 3), we analyzed the regulation of HO-1 in vascular
tissue in vivo. A dramatic increase in HO-1 mRNA
occurred in aortic tissue from rats receiving LPS compared with rats
receiving vehicle (Fig. 1A), and this increase in message was associated with an 8.9-fold increase in HO enzyme activity (Fig.
1B). HO-2 mRNA was not increased by LPS. The increase in HO-1 protein was present in vascular smooth muscle cells and
endothelial cells of both large (aorta, Fig. 2B) and small
(arterioles, Fig. 2, E and F) blood vessels. The
marked increase in HO enzyme activity by LPS (to a level of activity
comparable with that of the liver) would suggest that HO-1-generated CO
may contribute to the reduction in vascular tone during endotoxic
shock.
To clarify the role of HO-1 induction in endotoxic shock, we
administered ZnPP to rats that were made hypotensive by LPS. We used
ZnPP instead of other metalloporphyrins to inhibit HO activity because
ZnPP has been shown to be selective for HO over other microsomal
enzymes (14). Moreover, ZnPP in concentrations less than 50 µmol has
no inhibitory effect on NOS activity, and at a dose of 10 µmol it has
no effect on soluble guanylyl cyclase activity in vascular endothelial
cells (29). In our experiments, ZnPP given in a dose of 10 µmol/kg
abrogated the LPS-induced hypotension and it produced a significantly
higher mean arterial pressure in the LPS+ZnPP group (10 µmol/kg)
compared with the LPS alone group (Fig. 3). ZnPP at a dose of 1 µmol/kg prevented a further decrease in arterial pressure in rats
receiving LPS, and this response was less dramatic than in the
LPS-stimulated rats receiving 10 µmol/kg ZnPP. These data support the
importance of HO-1, and subsequently CO, in the hemodynamic compromise
of endotoxic shock.
Although our data support a detrimental role for vascular HO-1 in
endotoxic shock, a recent study proposed that further induction of HO-1
by hemoglobin may protect against the oxidative damage of endotoxemia
(33) by generating bilirubin (which has antioxidant properties; Ref.
34). These investigators demonstrated that the administration of HO
inhibitors at doses that decrease HO enzyme activity below basal levels
(SnPP and ZnPP, 50 µmol/kg) made rats more susceptible to LPS-induced
death (33). Unfortunately, the available inhibitors of HO activity do
not discriminate between HO-1 and HO-2. Thus, if HO-2 is an important
regulator of basal vascular homeostasis (29) and an important
neurotransmitter (35), we would speculate that inhibition of both HO-1
and HO-2 activity (similar to inhibition of both the constitutive and
inducible pathways of NOS; Ref. 36) would be harmful during endotoxic shock. Because of this hypothesis, we chose to administer lower doses
of ZnPP (10 and 1 µmol/kg) in rats after the onset of
endotoxin-induced shock to prevent complete inhibition of HO enzyme
activity. ZnPP given at the lower doses curtailed the LPS-induced
hypotension (Fig. 3). These data suggest that the beneficial
hemodynamic response to ZnPP in rats receiving LPS reflects an
inhibition of inducible HO activity.
Recent preliminary reports suggest that NO itself can induce HO-1
mRNA and protein expression in cell culture (26), and we have
demonstrated previously that IL-1 Our study demonstrates that HO-1-derived enzyme activity can be
up-regulated within vascular tissue by a pathophysiologic process,
endotoxemia, in vivo. In both large blood vessels (aorta) and small resistance vessels (arterioles), the increase in staining for
HO-1 localized to vascular smooth muscle cells and endothelial cells.
The advent of specific HO-1 antagonists, or an HO-1 gene deletion
animal, would allow us to gain more insight into the pathophysiologic
role of HO-1 in endotoxic shock. However, the marked induction of HO-1
enzyme activity by LPS within vascular tissue, and the beneficial
hemodynamic response to ZnPP in LPS-stimulated animals, would suggest
that HO-1 (and subsequently CO) contributes to the reduction in
vascular tone during endotoxic shock.
We extend our gratitude to Dr. Mu-En Lee and
Dr. Edgar Haber for helpful suggestions and continued enthusiasm and
support of our work. We also thank Dr. Mu-En Lee, Dr. Edgar Haber, Dr. Stella Kourembanas, and Yukari Perrella for critical reviews of the
manuscript; Thomas McVarish for editorial assistance; and Bonna Ith,
Donald Fletcher, Dorothy Zhang and Maureen Ibanez for technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
(1, 3). We have demonstrated
previously that IL-1
stimulates the inducible isoform of nitric
oxide synthase (NOS) and increases the production of NO in vascular
smooth muscle cells (4). NO is a labile, free radical gas that acts as
a potent vasodilator (5, 6). The importance of NO in the pathogenesis
of endotoxic shock has been emphasized by recent studies demonstrating
that mice carrying a disrupted inducible NOS gene have an attenuated hypotensive-response to LPS (7) and are resistant to LPS-induced death
(7, 8). However, the study by MacMicking and colleagues (7) also
suggested that an inducible NOS-independent pathway contributes to
LPS-induced hypotension and death, and we hypothesize that one
potential pathway involves heme oxygenase (HO).
Materials
20 °C. Recombinant human IL-1
(Collaborative Biomedical, Bedford, MA) was stored at
80 °C until use. ZnPP (Porphyrin
Products, Logan, UT) was dissolved in 0.1 N NaOH, then
immediately prior to administration this solution was neutralized with
0.1 N HCl as described (14). Because ZnPP has effects
unrelated to HO inhibition when exposed to light (15), we protected the
ZnPP from light during its preparation and use. Both the
L-arginine analogue
N
-nitro-L-arginine methyl ester
(L-NAME; Calbiochem, San Diego, CA) and
S-methylisothiourea sulfate (SMT; Sigma)
were dissolved in sterile water and stored at
20 °C until their
use.
80 °C for 4-12 h or
stored on phosphor screens for 2-4 h. To correct for differences in
RNA loading, the filters were washed in a 50% formamide solution at
80 °C and rehybridized with an 18 or 28 S ribosomal RNA probe.
Images were displayed, and radioactivity was measured on a
PhosphorImager running ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
(10 ng/ml) for 24 h. The
cells were subsequently lysed, and nuclei were isolated as described
(4, 18). Nuclear suspension (200 µl) was incubated with 0.5 mM each of CTP, ATP, and GTP and with 250 µCi of
32P-labeled UTP (3,000 Ci/mmol) (Du Pont NEN). The samples
were extracted with phenol/chloroform, precipitated, and resuspended at
equal counts/min/ml in hybridization buffer (9.7 × 106 cpm/ml). Denatured probes (1 µg) dot-blotted on
nitrocellulose filters were hybridized at 40 °C for 4 days in the
presence of formamide. cDNA for HO-1, HO-2, and
-actin genes
were used as probes.
1
cm
1 for bilirubin). HO enzyme activity was expressed as
nanomoles (nmol) of bilirubin formed/mg of protein/h. The protein
concentration was determined by a dye-binding assay (Bio-Rad).
(nitrite), by a standard method
(4, 23). The cells were cultured in Dulbecco's modified Eagle's
medium without phenol red for the experiments assessing nitrite
concentrations. The nitrite samples were normalized according to
protein concentration of the cultured cells (nmol/mg protein).
LPS Induces Vascular HO-1 mRNA and Enzyme Activity in
Vivo
Fig. 1.
Induction of HO-1 mRNA levels and HO
enzyme activity by LPS in aortic tissue in vivo.
A, conscious male Sprague-Dawley rats were injected with
vehicle (2 rats, lanes 1 and 2) or S. typhosa LPS (2 rats, lanes 3 and 4) at a
dose of 4 mg/kg intravenously. The rats were killed 9 h after LPS
or vehicle administration. Total RNA was extracted from the aortic
tissue, and Northern blot analysis was performed using 10 µg of total
RNA/lane. After electrophoresis the RNA was transferred to a
nitrocellulose filter, which was hybridized to a
32P-labeled rat HO-1 probe. The filter was also hybridized
with an 18 S ribosomal RNA probe to assess loading differences.
B, conscious male Sprague-Dawley rats were injected with
S. typhosa LPS (n = 2) at a dose of 4 mg/kg
intravenously or vehicle (n = 2). The rats were killed
9 h after LPS or vehicle administration and HO enzyme activity was
assessed in the aortic tissue. This experiment was performed in
duplicate, and HO enzyme activity is depicted as a mean ± S.D. of
the two experiments.
[View Larger Version of this Image (19K GIF file)]
Fig. 2.
Immunocytochemical localization of HO-1
expression in vascular tissue. Conscious male Sprague-Dawley rats
were injected with vehicle or S. typhosa LPS at a dose of 4 mg/kg intravenously. The rats were killed 9 h after vehicle or LPS
administration and perfused with 4% paraformaldehyde. The aortic
tissue was removed, and immunocytochemical staining was performed as
described (22, 25). Aortas from rats receiving vehicle (A;
magnification, ×600) or LPS (B; magnification, ×600) were
stained using a rabbit anti-HO-1 antibody. In addition, adventitial
arterioles (vessels less than 300 µm in diameter) from rats receiving
vehicle (C and D; magnification, ×600) or LPS
(E and F; magnification, ×600) were examined for HO-1. The brown color represents positive staining for HO-1.
In the arteriole from the rat receiving LPS (E), arrow
EC points to an endothelial cell and arrow SMC points
to smooth muscle cells staining positive for HO-1.
[View Larger Version of this Image (90K GIF file)]
Fig. 3.
Effect of ZnPP on LPS-induced hypotension in
male Sprague-Dawley rats. LPS group (n = 4)
received LPS (4 mg/kg intra-arterially) and saline vehicle in place of
ZnPP. The LPS+ZnPP groups (n = 4 in each group)
received LPS (4 mg/kg intra-arterially) followed by ZnPP (either 10 or
1 µmol/kg intraperitoneally). The ZnPP group (n = 4)
received ZnPP (10 µmol/kg intraperitoneally) and saline vehicle in
place of LPS. After an initial 25% decrease in mean arterial pressure
(corresponding to time point 0) in the rats receiving LPS (LPS group
and LPS+ZnPP groups), ZnPP (or saline vehicle) was administered and
mean arterial pressure was monitored over the next 90 min. The values
represent mean ± S.E. *, significant decrease in mean arterial
pressure in LPS and LPS+ZnPP (10 µmol/kg and 1 µmol/kg) groups
versus their own base lines, and versus ZnPP (10 µmol/kg) group. , significant decrease in LPS group versus LPS+ZnPP (10 µmol/kg) and ZnPP (10 µmol/kg)
groups. §, significant decrease in LPS group versus all
other groups. Inset, total RNA was extracted from the aortic
tissue of rats at base-line level or after LPS stimulation
corresponding to time point 0. Northern blot analysis was performed
using 10 µg of total RNA/lane. After electrophoresis the RNA was
transferred to a nitrocellulose filter, which was hybridized to a
32P-labeled rat HO-1 probe. The filter was also hybridized
with an 18 S ribosomal RNA probe to assess loading differences. The signal intensity of each RNA sample hybridized to the HO-1 probe was
divided by that hybridized to the 18 S probe, and the normalized intensities were then plotted as -fold increase in HO-1 mRNA
from base-line levels.
[View Larger Version of this Image (25K GIF file)]
Induces HO-1, but Not HO-2, mRNA in Vascular Smooth
Muscle Cells in Vitro
in cultured RASMC. Northern blot analyses were
performed with total RNA from RASMC exposed to either vehicle or
IL-1
. The blots were then hybridized to HO-1 and HO-2 cDNA
probes. A representative Northern blot of a time-course experiment of
HO-1 stimulation by IL-1
(10 ng/ml, dose promoting maximal
induction) is presented in Fig. 4A. In
contrast to the more rapid in vivo induction (within 2 h), IL-1
did not increase HO-1 mRNA until 4 h after
stimulation in vitro, and peak induction occurred after
24 h. The time course of HO-1 mRNA stimulation by IL-1
,
including additional samples extending the stimulation to 48 h, is
graphically illustrated in Fig. 4B. Twenty-four hours after
the administration of IL-1
, HO-1 mRNA increased by 5.8-fold
compared to vehicle. The induction of HO-1 mRNA by IL-1
decreased to 2.8-fold after 48 h. The message for HO-2, in
contrast to HO-1, varied minimally after treatment with IL-1
(Fig.
4C).
Fig. 4.
Time course of HO-1 and HO-2 mRNA
induction by IL-1 in RASMC. A, RASMC were exposed to
IL-1
(10 ng/ml) and total RNA was extracted from the cells at the
indicated times. RNA was also extracted from cells receiving a vehicle,
but not IL-1
, at the indicated times. Northern blot analyses were
performed using 5 µg of total RNA/lane. After electrophoresis the RNA
was transferred to nitrocellulose filters, which were hybridized to 32P-labeled rat HO-1 probe. The filters were also
hybridized with a 32P-labeled oligonucleotide probe
complementary to 18 S ribosomal RNA to assess loading differences.
B, the signal intensity of each RNA sample hybridized to the
HO-1 probe was divided by that hybridized to the 18 S probe, and the
normalized intensities were then plotted as a percentage of vehicle.
Additional samples extending the IL-1
stimulation to 48 h were
included. C, the above filters were also hybridized with a
32P-labeled rat HO-2 probe, and the signal intensity of
each RNA sample hybridized to the HO-2 probe was divided by that
hybridized to the 18 S probe, and the normalized intensities were then
plotted as a percentage of vehicle.
[View Larger Version of this Image (23K GIF file)]
Increases the Rate of HO-1 Gene Transcription
increases HO-1 mRNA in
vascular smooth muscle cells, we performed experiments to assess the
transcriptional rate and mRNA stability of HO-1. Nuclear run-on
experiments showed a 5-fold increase in the rate of HO-1 gene
transcription after 24 h of IL-1
stimulation (Fig.
5A). Stability of HO-1 message experiments,
as determined by measuring mRNA levels in the presence of the
transcription inhibitor actinomycin D, demonstrated a mRNA
half-life of 1.3 h after exposure to vehicle (Fig. 5B).
The half-life of HO-1 mRNA was not prolonged by IL-1
. These
experiments demonstrate the increase in HO-1 mRNA levels by IL-1
stimulation is the result of an increase in HO-1 gene transcription.
IL-1
did not alter the transcriptional rate (Fig. 5A) or
the half-life (data not shown) of HO-2 mRNA.
Fig. 5.
Effect of IL-1 on HO-1 transcriptional
rate and mRNA stability. A, RASMC were either stimulated
with IL-1
or vehicle for 24 h. Nuclei were then isolated from
the RASMC, and in vitro transcription was allowed to resume
in the presence of [
-32P]UTP. Equal amounts of
32P-labeled, in vitro transcribed RNA probes
from each group were hybridized to 1 µg of denatured HO-1,
-actin,
and HO-2 cDNA that had been immobilized on nitrocellulose filters.
The bar graph represents the signal intensity of HO-1
normalized by
-actin, and the transcriptional rate was plotted as a
percentage of vehicle. B, RASMC were stimulated with IL-1
(10 ng/ml) or vehicle for 12 h, then actinomycin D (5 µg/ml) was
administered to the RASMC. Total RNA was extracted from the RASMC at
the indicated times after administration of actinomycin D. Northern
blot analyses were performed using 5 µg of total RNA/lane. After
electrophoresis the RNA was transferred to nitrocellulose filters,
which were hybridized to 32P-labeled HO-1 and 18 S probes.
To correct for differences in loading, the signal intensity of each RNA
sample hybridized to the HO-1 probe was divided by that hybridized to
the 18 S probe. The normalized intensity was then plotted as a
percentage of the 0-h value against time (in log scale).
[View Larger Version of this Image (17K GIF file)]
Is Not Prevented by
Inhibitors of NOS
(10 ng/ml) for 24 h in
the presence or absence of L-NAME (10
3
M). L-NAME did not inhibit IL-1
-induced HO-1
mRNA induction (Fig. 6A). This study was
also performed using a more specific inhibitor of inducible NOS (27),
SMT. SMT (10
4 M) also had no effect on the
induction of HO-1 mRNA by IL-1
(Fig. 6A). To make
sure that NO production was inhibited by L-NAME and SMT, we
measured a stable product of NO oxidation,
NO2
(nitrite) (23), in these
experiments. The doses of L-NAME (10
3
M) and SMT (10
4 M) were chosen
after performing dose-response experiments to select the minimum dose
of each agent necessary to inhibit the accumulation of nitrite in the
culture media (data not shown). Both L-NAME and SMT
significantly inhibited the accumulation of nitrite (Fig.
6B); however, they had no effect on the induction of HO-1
mRNA levels. These studies demonstrate that the induction of HO-1
message by IL-1
occurs through a NO-independent pathway.
Fig. 6.
Effect of NOS inhibitors on the induction of
HO-1 mRNA by IL-1 in RASMC. A, RASMC were exposed to
no stimulus (control), IL-1
(10 ng/ml), IL-1
(10 ng/ml) plus
L-NAME (10
3 M),
L-NAME (10
3 M) alone, IL-1
(10 ng/ml) plus SMT (10
4 M), or SMT
(10
4 M) alone. Total RNA was extracted from
the cells after 24 h of stimulation. Northern blot analyses were
performed using 10 µg of total RNA/lane. After electrophoresis the
RNA was transferred to nitrocellulose filters, which were hybridized to
32P-labeled HO-1 and 28 S probes. B, RASMC were
exposed to no stimulus (control), IL-1
(10 ng/ml), IL-1
(10 ng/ml) plus L-NAME (10
3 M),
L-NAME (10
3 M) alone, IL-1
(10 ng/ml) plus SMT (10
4 M), or SMT
(10
4 M) alone. Extracellular nitrite
accumulation was assayed from the culture media after 24 h of
stimulation. NO production was expressed as nanomoles/mg of protein.
The values represent the mean ± S.D. (n = 3).
[View Larger Version of this Image (20K GIF file)]
stimulates an increase in NO
production through the inducible NOS pathway in vascular smooth muscle
cells in vitro (4). Thus, to determine if the induction of
HO-1 mRNA by IL-1
occurs indirectly through an increase in NO
production, we stimulated vascular smooth muscle cells with IL-1
in
the presence of two different NOS inhibitors. Neither L-NAME nor SMT (both of which inhibited the accumulation of
NO) were able to impede the induction of HO-1 mRNA by IL-1
(Fig. 6). These data suggest the induction of HO-1 mRNA by IL-1
occurs through a NO-independent pathway in vascular smooth muscle cells.
*
This work was supported in part by a grant from the
Bristol-Myers Squibb Pharmaceutical Research Institute. 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.
Recipient of a National Research Service Award from the National
Institutes of Health.
Supported by Grant KO8HL03194 from the National Institutes of
Health. To whom correspondence should be addressed: Cardiovascular Biology Laboratory, Bldg. 2, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-2273; Fax: 617-432-2980; E-mail: perrella{at}cvlab.harvard.edu.
1
The abbreviations used are: LPS,
lipopolysaccharide; HO, heme oxygenase; IL-1, interleukin-1
;
ZnPP, zinc protoporphyrin IX; NOS, nitric oxide synthase;
L-NAME,
N
-nitro-L-arginine methyl ester;
SMT, S-methylisothiourea sulfate; RASMC, rat aortic smooth
muscle cells.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.