Carbon monoxide overproduced by heme oxygenase-1 causes a reduction
of vascular resistance in perfused rat liver
Yoshiyuki
Wakabayashi1,
Rina
Takamiya1,
Akira
Mizuki2,
Takanori
Kyokane1,
Nobuhito
Goda1,
Tokio
Yamaguchi3,
Shinji
Takeoka4,
Eishun
Tsuchida4,
Makoto
Suematsu1, and
Yuzuru
Ishimura1
1 Department of Biochemistry,
School of Medicine, Keio University,
2 Department of Medicine,
Saisei-kai Central Hospital,
4 Department of Polymer Chemistry,
Waseda University, and
3 Department of Biochemical
Genetics, Medical Research Institute, Tokyo Medical and Dental
University, Tokyo 160, Japan
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ABSTRACT |
This study aimed to examine whether livers
overexpressing heme oxygenase (HO)-1 could alter the vascular
resistance through the vasorelaxing action of carbon monoxide (CO). The
relationship among HO-1 expression, CO generation, and the vascular
resistance was assessed in perfused rat livers pretreated with hemin,
an inducer of HO-1. At 18 h after the hemin treatment, livers displayed marked increases in HO-1 expression in hepatocytes and venous CO flux
and a reduction of the basal resistance. The reduction of the
resistance in hemin-treated livers was canceled by administration of
oxyhemoglobin, a reagent trapping both CO and nitric oxide (NO), but
not by methemoglobin, which captures NO but not CO. Liposome-encapsulated oxyhemoglobin, which cannot access the space of
Disse, did not cause vasoconstriction. Furthermore, these livers became
less sensitive to endothelin-1, a vasoconstrictive peptide, than the
untreated controls through mechanisms involving CO. On the other hand,
at 12 or 24 h after the treatment when the HO-1 induction was not
accompanied by CO overproduction, neither a decrease in the basal
resistance nor vascular hyporeactivity to endothelin-1 was
observed. These results suggest that CO overproduced in
the extrasinusoidal compartment is a determinant of the HO-1-mediated vasorelaxation in the liver.
heat shock protein 32; cytochrome
P-450; hepatic sinusoids; hemoglobin; methemoglobin
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INTRODUCTION |
HEME OXYGENASE (HO) is an enzyme responsible for the
degradation of protoheme IX to form biliverdin, free iron, and carbon monoxide (CO). The enzyme exists in three isoforms: HO-1 (28), HO-2
(15), and HO-3 (16). Among them, HO-1, which is identical to heat shock
protein 32, is induced by a variety of stimuli such as cytokines, heavy
metals, and oxidants (14). This inducible isoform can also be
upregulated by protoheme IX (iron protoporphyrin IX) (15), the
substrate for HO. Approximately 70% of the heme degraded in the body
is derived from hemoglobin from senescent erythrocytes (14).
Liver is one of the major organs in which the heme
molecules are oxidatively degraded by HO, and its capacity to decompose the heme is increased by the HO-1 induction. Recent studies also revealed that induction of HO-1 occurred in the liver in response to
endotoxemia (6) and hemorrhagic shock (2). The hepatic HO-1 expression
could also be markedly elevated when amounts of free heme molecules in
circulation are acutely increased. Such circumstances involve massive
hemolysis by sepsis (27), excessive blood transfusion, and splenectomy
(35).
Recently, several lines of evidence indicated that CO produced by the
HO reaction contributes to regulation of cell functions by activating
soluble guanylate cyclase (4, 36) or by hyperpolarizing membrane
potentials through the mechanisms involving stimulation of potassium
channels (21). Although these previous studies suggest a possible role
of the endogenous CO-generating system in regulation of cell and organ
functions, the relationship between actual CO amounts released through
the endogenous origins and resultant influences on the cell function
have not yet been fully investigated. We have shown in the liver that
CO endogenously generated by the HO reaction is necessary to maintain
sinusoidal vessels in a relaxing state (32, 33). Administration of zinc protoporphyrin IX (ZnPP), a potent inhibitor of HO, or of free oxyhemoglobin (HbO2), a trapping
agent of CO, markedly increased the vascular resistance concurrent with
sinusoidal constriction. HbO2 can
diffuse across the fenestrated endothelium of the liver into the space
of Disse and thereby entrap CO in the extrasinusoidal space.
Considering that hepatocytes that constitutively express HO-2 are the
major sites for CO generation, these results suggested that CO
generated by HO-2 serves as a regulator of sinusoidal tone under
ordinary conditions (10). Such a physiological role of CO
constitutively generated by HO-2 raised a question as to whether the
liver undergoing stress conditions alters its vascular function through
overproduction of CO as a consequence of the HO-1 induction. The
present study was thus undertaken to demonstrate time history and
topographic distribution of the HO-1 expression, their relationship
with quantitative evaluation of local CO generation, and its functional
link to vascular tone.
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MATERIALS AND METHODS |
Reagents.
Anti-rat HO-1 and HO-2 antibodies were prepared and purified as
described elsewhere (10). ZnPP was purchased from Aldrich Chemical
(Milwaukee, WI). Other reagents used were purchased from Sigma Chemical
(St. Louis, MO) unless otherwise mentioned.
Preparation of hemoglobin and liposome-encapsulated hemoglobin
vesicles.
To examine the role of endogenously generated CO in the regulation of
vascular tone in the liver, free and liposome-encapsulated hemoglobins,
hereafter designated as free Hb and HbV, respectively, were used as a
tool to trap CO in and around the sinusoidal space. Free Hb, HbV, and
methemoglobin (metHb) were prepared using outdated human erythrocytes
as described previously (23, 24). metHb was used as a tool to scavenge
nitric oxide (NO) but not CO. The mean value of the diameter of HbV
used in the present study was ~0.25 µm. Previous studies (22)
revealed that, when administered into the systemic circulation, HbV
with this size was partly captured in tissue macrophages including
Kupffer cells but did not enter the hepatic parenchyma. On the other
hand, free Hb was readily trapped by the parenchymal cells because of
the presence of fenestration in sinusoidal endothelium, the diameter of
which ranges from 0.10 to 0.15 µm (18, 37). The oxygen-binding
affinity exhibited no substantial differences between HbV and free Hb,
as described previously (23). Both free Hb and HbV were used for
experiments within 7 days after the preparation procedures.
Animal preparation and hemin treatment.
Male Wistar rats (260-280 g) were obtained from Saitama Animal
Laboratory (Saitama, Japan). All animals were allowed free access to
laboratory chow and tap water. Rats were pretreated with hemin via an
intraperitoneal injection (40 µmol/kg). They were anesthetized
intraperitoneally with pentobarbital sodium (50 mg/kg), and the common
bile duct was cannulated by a polyethylene (PE)-10 catheter. The livers
were excised and perfused with Hb- and albumin-free Krebs-Henseleit
bicarbonate-buffered solution (pH 7.4, 37°C) gassed 95%
O2-5%
CO2 containing 30 µM
taurocholate as described previously (25, 40). The perfusate was pumped through the liver with a peristaltic pump at a constant rate of 4.0 ml · min
1 · g
liver weight
1 in a single
pass mode while the inlet perfusion pressure was monitored
(40).
Experimental protocols.
Five main protocols were employed to compare the differences in the
vascular resistance between the control and the hemin-treated livers.
Livers in the first, second, and third groups were treated with
HbO2,
HbV-O2, and metHb, respectively.
Concentrations of the Hb derivatives were all 0.5 g/dl and were usually
perfused for 10 min. In the fourth group, 1 µM ZnPP, an HO inhibitor,
was applied to the perfusate. In the fifth group, the same
concentration of ZnPP was perfused with CO supplemented at 4 µM, the
concentration that can sufficiently compensate the CO suppression by
ZnPP. Effects of these reagents on the vascular resistance were also
examined in livers pretreated with endothelin-1 (ET-1); in these
experiments, the reagents were added to the perfusate for 5 min
followed by the infusion of 1 nM ET-1 for 1 min. The dose of ET-1 was
estimated to be ~30 pmol/min for 1 min. This dose of ET-1 was
reported to evoke site-specific constriction of microvessels including
portal venules and sinusoids but not of larger portal veins (11, 34). We confirmed that no significant reduction of bile flow was observed after injecting this dose of ET-1 (data not shown). This observation convinced us that there was little involvement if any of
vasoconstriction at the level of the portal veins. In some experiments,
desired concentrations of
N
-nitro-L-arginine methyl ester
(L-NAME) were added to the
perfusate to suppress endogenously generated NO. ET-1 and
L-NAME were purchased from
Sigma. In all experiments, bile samples were collected every 5 min to
monitor the bile output.
Digital microfluorography of sinusoids in perfused liver.
To examine alterations in the diameter of sinusoids, digital
microangiography was carried out in perfused rat livers by using real-time laser confocal microscopy as described elsewhere (10, 29).
Briefly, the surface of the perfused liver was observed through an
inverted-type intravital microscope assisted by a line-scan laser
confocal imager (Insight/TMD300, Meridian Far East, Tokyo, Japan), and
the hepatic microcirculation was visualized by a 10-bit color chilled,
intensified charge-coupled device camera (C5810-01, Hamamatsu
Photonics, Hamamatsu). This system allowed us to acquire the confocal
fluorescence images at a video-rate speed (30 frames/s); images were
optically sliced to the desired width, which ranged from 1 to 20 µm. To visualize the hepatic sinusoidal vessels, 100 µl of
1% of FITC-dextran solution were injected transportally every 2 or 5 min after the start of experiments, and the microvasculature was
visualized by epi-illumination at 488 nm using an argon laser light
source. The width of the confocal plane along the
z-axis was set up to 2 µm to collect
the fluorescent images.
Determinations.
Rat liver homogenates were subjected to SDS-PAGE, transferred to a
Millipore Immobilon polyvinylidene difluoride transfer membrane, and
then served as samples for Western blot analysis, as described
previously (10). HO-1 and HO-2 proteins were visualized using specific
antibodies against rat HO-1 and HO-2, namely, GTS-1 and
GTS-2, respectively (10).
Concentrations of CO in the effluent of perfused liver was determined
by detecting ferrous CO complex of myoglobin spectrophotometrically (32). Bilirubin IX
concentrations in bile samples were determined by
ELISA using the monoclonal antibody 24G7, which recognizes both
conjugated and unconjugated bilirubin IX
fractions as described previously (39, 41). All experimental procedures for bilirubin measurements were carried out under subdued light.
Hepatic microsomal cytochrome P-450
contents were measured by the spectrophotometric method of Omura and
Sato (19). Activities of HO in liver tissues were determined by
measuring the bilirubin formation as described elsewhere (42). Protein
was determined according to the method of Lowry et al. (13).
Immunohistochemistry of liver HO-1 and HO-2.
Frozen sections (7 µm thick) were prepared from rat livers. After
fixation with acetone, the sections were treated with avidin, biotin,
and normal horse serum to minimize the nonspecific staining. These
tissues were incubated with monoclonal antibodies to GTS-1 or GTS-2,
dissolved in 1% BSA-PBS at a final concentration of 1 µg/ml, for at
least 2 h at 25°C. After several washes with PBS, the sections were
stained with anti-mouse IgG for 1 h (Vectastain Elite ABC kit, Vector,
CA). To prevent endogenous peroxidase reactions, the samples were
pretreated with 0.3%
H2O2
in cold methanol for 30 min and subsequently incubated with avidin and
horseradish peroxidase-conjugated biotin for 30 min. Finally, 0.1 mg/ml
of 3,3'-diaminobenzidine tetrahydrochloride were applied to
sections for 5 min. The sections were counterstained with methyl green after fixation with 20% formaldehyde for 20 min, and slides were coverslipped with aqueous mounting medium. The immunohistochemical staining patterns were examined using the samples collected from at
least three individual rats. To confirm the specificity of immunohistochemical localization, antibodies preabsorbed with an excess
of adequate antigens in advance were used.
Statistical analyses.
All data in the present study are expressed as means ± SE of
measurements. Differences in the mean values among the groups were
analyzed by one-way ANOVA combined with Scheffé's-type multiple comparison test. P values <0.05 were
considered statistically significant.
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RESULTS |
HO-1 expression and CO generation in the hemin-treated liver.
Figure 1 illustrates time history of the
HO-1 expression in the liver of rats treated with an intraperitoneal
injection of 40 µmol/kg hemin. As shown in Fig.
1A, the HO activity in the tissue
increased time dependently and reached a maximum at 12 h, plateaued
until 18 h, and then was followed by a reduction at 24 h. Results for
the immunoreactive HO-1 protein also displayed a time course similar to
the results for HO activity, as assessed by Western blot analysis (Fig.
1B). Because the expression of HO-2,
as assessed by Western blot analysis (data not shown), showed no
significant changes during the 18-h period, these results indicate that
an increase in the HO activity is attributable to the increase in HO-1
protein expression. On the other hand, the time course of the CO flux
in the venous effluents exhibited quite different pictures, as
indicated in Fig. 1C. At 6 h, the flux
displayed no significant elevation, in contrast to the HO-1 activity,
which was 2.7-fold greater than that in the control, as shown in Fig. 1A. However, at 12 h, when the
enzyme activity reached a maximum level, the CO flux did not show any
significant changes. However, at 18 h, the CO flux exhibited a marked
elevation, being as much as 4.5-fold greater than the control flux. The
elevation was markedly repressed at 24 h, at which time HO-1 protein
expression exhibited a fall nearly to control levels (Fig.
1B).

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Fig. 1.
Time history of the heme oxygenase (HO) activity, the protein
expression of HO-1, and carbon monoxide (CO) generation in livers from
the hemin-treated rats.
A-C:
temporal alterations in the microsomal enzyme activity, Western
blotting analysis, and flux of CO measured in the venous samples
collected from the perfused liver, respectively.
D: time course of contents of
cytochrome P-450 per mg microsomal
protein. Note that a marked increase in the CO flux occurred at 18 h,
whereas the enzyme expression and activity reached a maximum level at
12 h. Data in A,
C, and
D represent means ± SE of
measurements from 4-5 separate livers.
* P < 0.05 compared with
control values measured at time 0.
P < 0.05 compared with
values at 12 h. # P < 0.05 compared with values at 18 h.
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Differences in time history between the HO-1 induction and CO
generation led us to examine alterations in amounts of the substrate available for the HO reaction. We therefore examined contents of
cytochrome P-450, a major heme protein
that could provide protoheme IX as a substrate for HO. As seen in Fig.
1D, contents of cytochrome P-450 increased gradually during the
initial 12 h. At 18 h, however, the liver displayed as much as a 45%
decrease in microsomal cytochrome P-450 contents compared with that
treated for 12 h.
Reduction of the basal vascular resistance in the CO-overproducing
liver.
We then investigated whether the liver undergoing the HO-1 induction
alters its organ functions, such as vascular resistance and bile
formation. As seen in Table 1, these
functional parameters exhibited no significant changes in the 12-h
hemin-treated liver. However, the liver exposed to the 18-h hemin
treatment displayed a 13% reduction of the basal vascular resistance.
Bile output was elevated concurrently with reduction of the resistance,
showing a 10% increase vs. control values. Although small, these
changes were statistically significant. We also examined differences in the biliary flux of bilirubin IX
as an index of the HO-mediated heme
degradation, which became 4.8-fold greater at 18 h than that shown in
the controls, the value being stoichiometric to the increase in the
venous CO flux. These results indicate that alterations in the vascular
resistance and bile output are the events occurring in concert with the
overproduction of CO.
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Table 1.
Changes in the baseline vascular resistance, bile output, and biliary
flux of bilirubin IX in the hemin-challenged rat liver
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Intralobular distribution of HO-1 expression in the hemin-pretreated
liver.
Figure 2 illustrates immunohistochemical
analysis using a monoclonal antibody against HO-1. In the control
liver, HO-1 was densely stained in cells located in nonparenchymal
regions, whereas only little staining was observed in parenchymal cells
(Fig. 2A). The sites of the HO-1
expression corresponded to the distribution of Kupffer cells as
described previously (10). In contrast, HO-2 was mainly observed in the
parenchymal cells (data not shown) (10). After the 18-h hemin
treatment, HO-1 expression was demonstrated not only in Kupffer cells
but was also prominent in hepatocytes throughout the entire lobule
(Fig. 2B). On the other hand, HO-2 expression in the hemin-treated liver displayed no detectable changes
between the control and the 18-h hemin-treated livers (data not shown).
Together with the data from Fig. 1,
A-C,
these results suggest that the overproduction of CO in the livers of hemin-treated rats is ascribable to a marked induction of HO-1 in
parenchymal cells and to the increase in availability of cytochrome P-450-derived heme.

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Fig. 2.
Immunohistochemical determination of HO-1 expression in the
hemin-pretreated rat livers. The 7-µm-thick frozen sections of rat
livers were immunostained with GTS-1.
A and
B: samples collected from the control
and 18-h hemin-treated livers, respectively. Bar = 100 µm. P and C
denote portal and central venules, respectively.
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Effects of HbO2 and ZnPP on vascular
resistance.
We investigated whether reduction of vascular resistance observed at 18 h of treatment is indeed controlled by mechanisms involving CO. Figure 3 illustrates
alterations in the baseline vascular resistance and the sinusoidal
caliber in response to administration of 0.5 g/dl
HbO2, a CO-trapping reagent.
Figure 3A shows that, immediately
after the start of administration, the vascular resistance exhibited a
marked elevation, being 33% greater than the baseline level. When
HbO2 was eliminated from the
perfusion circuit at 10 min after the administration, the increased
resistance decreased back to the baseline level within 20 min, showing
that the HbO2-induced elevation of
resistance is a reversible event. On the other hand, metHb, a reagent
that could scavenge NO but not CO, did not alter the vascular
resistance. Furthermore, administration of NO synthase inhibitors such
as L-NAME and aminoguanidine did
not increase the resistance (data not shown). These results suggest
that CO rather than NO plays a major role in lowering the baseline
vascular resistance under the current experimental conditions. To
specify the role of CO generated in intra- and extravascular
compartments, effects of liposomal encapsulation of
HbO2 on the resistance were also
examined. Administration of HbV-O2
induced no significant changes in vascular resistance, indicating that
CO generated in the extravascular space is important in lowering the
baseline vascular resistance.

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Fig. 3.
Effects of oxyhemoglobin (HbO2),
methemoglobin (metHb), and liposome-encapsulated hemoglobin (HbV) on
the vascular resistance in perfused rat liver exposed to the 18-h hemin
treatment. In A, open triangles denote
data collected from the HO-1-induced liver receiving no hemoglobin
derivatives. and , Data from livers receiving
HbO2 and HbV, respectively. ,
Data from metHb-treated liver. Results are means ± SE of 6 separate
experiments, expressed as percentages vs. basal vascular resistance in
the 18-h hemin-treated liver (see Table 1).
* P < 0.01 compared with
control ( ). B: time
course of alterations in the sinusoidal caliber during
HbO2 administration. Twenty
individual sinusoids in a representative liver preparation were chosen
for measurements, and their changes in caliber were determined before
and 2, 5, 10, 15, and 20 min after the start of the
HbO2 administration.
HbO2 was eliminated from the
perfusion circuit at 10 min. Bold lines indicate means ± SE of the
20 measurements in the representative experiment.
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Figure 3B illustrates alterations in
the sinusoidal caliber determined by FITC-dextran-assisted
microangiography during HbO2 administration. The vascular components responsible for the
HbO2-induced elevation of
resistance appear to involve sinusoidal vessels. In good agreement with
our previous observation in the control perfused liver (31), a large
variation of the caliber not only among different sinusoids but among
different sites in the same sinusoidal segment was observed in the 18-h
hemin-treated livers (data not shown). In response to the addition of
HbO2 to the perfusate, sinusoids
of these livers displayed a significant decrease in the caliber as
early as 2 min after the start of administration, showing about a 20%
decrease. The HbO2-induced changes
in sinusoids were reversed with elimination of
HbO2, suggesting this event is reversible.
A similar elevation of the vascular resistance was also observed after
administration of an HO inhibitor (Figure
4). ZnPP (1 µM) abolished the
hemin-induced reduction and caused further elevation of
the vascular resistance (about a 20% increase compared with that in
the control liver). The ZnPP-induced elevation was significantly
lowered by coperfusion with 4 µM CO, although not completely
abolished. These results suggest that, in the hemin-treated liver, the
low vascular resistance observed is caused at least in part by CO
endogenously generated by HO.

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Fig. 4.
Effects of zinc protoporphyrin IX (ZnPP), an inhibitor of HO, on
baseline vascular resistance in the 18-h hemin-pretreated rat liver
(H18). Baseline vascular resistance was plotted as relative values vs.
those in control. Namely, the value 100 indicates 2.42 ± 0.13 cmH2O · g
liver · min · ml 1.
ZnPP was administered at final concentrations of 1 mM. When added, 4 µM CO was applied to the perfusate 5 min before ZnPP administration.
Data are means ± SE of measurements from 6-9 separate livers.
* P < 0.05 compared with
control value. P < 0.05 compared with values measured in 18-h hemin-treated liver.
# P < 0.05 compared with
values in 18-h hemin-treated liver perfused in the presence of 1 µM
ZnPP.
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Overproduction of CO diminished ET-1-elicited sinusoidal
constriction.
The above studies with different Hb derivatives showed that the
reduction of basal vascular resistance in the HO-1-induced liver is
attributable to relaxation of microvessels by overproduced CO. This
finding tempted us to examine whether the CO overproduction can
predispose sinusoids to actively relax even under stimulated conditions. To this end, we applied ET-1 to constrict microvessels. This vasoconstrictive peptide can increase sinusoidal tone at a dose
that does not evoke constriction of larger portal veins (34) but
elicits that of microvessels involving portal venules (11) and/or
sinusoids (3). As shown in Fig. 5, 1.0 nM
but not 0.5 nM ET-1 evoked an increase in the vascular resistance. In
contrast, the liver undergoing the 18-h hemin treatment displayed a
45% decrease in the ET-1-elicited elevation of the resistance, whereas
that exposed to the 12-h treatment exhibited no changes. We did not
observe interlobular heterogeneity of the perfusion in these livers as
assessed by the transportal injection of trypan blue at the end of
experiments (data not shown). In addition, during the ET-1
administration, the bile output did not show any detectable decrease.
On the other hand, administration of 5 nM ET-1 evolved a twice greater
increase in the resistance, which coincided with a marked reduction of
the bile output and an interlobular heterogeneity in the perfusion.
Considering that greater doses of ET-1 are known to evoke cholestatic
changes in parallel with a marked constriction of portal veins (3, 34),
these results suggest that the increase in vascular resistance elicited
by ET-1 at <1 nM is ascribable to vasoconstriction of
microvasculature, including portal venules and/or sinusoids, rather
than to vasoconstriction of larger portal veins. On the basis of these
data, we compared the differences between the control and the 18-h
hemin-treated livers in the ET-1-induced vascular response at 1 nM.

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Fig. 5.
Differences in responses of endothelin-1 (ET-1)-induced elevation of
the vascular resistance in HO-1-induced livers. ET-1 was administered
transportally at concentrations ranging from 0.1 to 1 nM for 1 min.
Values of change in ( ) vascular resistance were determined by
calculating the difference between the resistance measured at 4 min
after ET-1 injection and that measured before injection. and ,
Data collected from control and 12-h hemin-treated livers,
respectively. , Data from 18-h hemin-treated livers. Note that the
liver undergoing 18-h hemin treatment specifically decreased its
vasoconstrictive sensitivity to ET-1.
* P < 0.05 compared with
control value. P < 0.05 compared with values measured in 18-h hemin-treated liver.
# P < 0.05 compared with
values in 18-h hemin-treated liver perfused in the presence of 1 µM
ZnPP. Inset: effects of
N -nitro-L-arginine methyl ester
(L-NAME), a nitric oxide
synthase inhibitor, on ET-1-induced elevation of resistance. and
, data collected from 12-h hemin-treated livers in the absence or
presence of 1 mM L-NAME,
respectively. Closed and open arrows indicate time points when
L-NAME and ET-1 were
administered to the perfusate, respectively. Mean value of the
vascular resistance induced by 1 nM ET-1 in 18-h hemin-treated liver
(see in main panel) was expressed as 100%. Data are means ± SE
of 5 separate experiments.
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Because ET-1 is known to stimulate NO generation from endothelial cells
and to help reduce the ET-1-elicited increase in the vascular tone
(26), we inquired whether the hemin-treated liver could display greater
sensitivity of the NO-mediated relaxation in response to the ET-1
administration and thereby exhibit smaller vasoconstrictive responses
than the ordinary liver. Figure 5, inset, illustrates the effects of
administration of 1 mM L-NAME on
the ET-1-induced elevation of the vascular resistance in the hemin-treated liver. The increase in the vascular resistance by ET-1
was not significantly altered by the presence of the NO synthase inhibitor. These results suggest that, at least under the current experimental conditions, the decreasing sensitivity of the resistance in response to the ET-1 stimulation observed at 18 h is unlikely to be
NO dependent.
Table 2 displays effects of ZnPP, an HO
inhibitor, on resistance in the HO-1-induced liver under the
ET-1-stimulated conditions. The ET-1-induced elevation of the
resistance was significantly reduced in the 18-h treated livers
compared with that in the control. In the presence of 1 µM ZnPP in
the perfusate, the ET-1-induced response was not reduced and further
increased to 120% of the control value. The ZnPP-induced changes were
attenuated in part by supplement of 4 µM CO. These results suggest
that the ET-1-induced elevation of the resistance was reduced in part
by CO overproduced by HO in the 18-h hemin-treated liver.
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Table 2.
Effects of ZnPP and CO on the ET-1-induced changes in the vascular
resistance in the 18-h hemin-treated liver
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DISCUSSION |
Previous studies suggested that, under various disease conditions, HO-1
is induced and thereby causes alterations in cell and organ functions
(1, 2, 5, 20). However, outcome of the functional alterations on the
enzyme induction was not sufficiently elucidated in these previous
studies. The present study provides substantial evidence that CO
overproduced by the inducible HO serves as an endogenous vasodilating
factor that alters the hepatic vascular resistance. Quantitative
determination of CO as a terminal product of the HO pathway disclosed
that, under our experimental conditions, actual
generation of the product rather than amounts of the enzyme expressed
in the tissue dictated changes in the vascular function.
Immunohistochemical analysis illustrates that both parenchymal and
nonparenchymal cells constitute major cellular components responsible
for the regional CO production. Furthermore, entrapment of CO generated
in situ by HbO2 as well as enzyme
inhibition by ZnPP elicited marked elevations of resistance, suggesting
that CO is a determinant for the decreased vascular resistance in the
HO-1-induced liver, at least under the current experimental conditions
using isolated perfused preparations.
We reported that CO generated by the constitutive isozyme of HO, i.e.,
HO-2, plays a major role in maintaining sinusoids in a relaxing state
under ordinary conditions (10, 32). The biological significance of the
HO-1-mediated vasorelaxing mechanism depicted in this study appears to
be distinct from that of the HO-2-mediated one, although these two
isozymes share the same reaction product as the vasorelaxing reagent.
Because of the greater Michaelis-Menten constant value for protoheme IX
(16), an induction of HO-1 under disease conditions that induce
overloading of heme iron observed in sepsis and hemolysis has been
thought to play an important role in normalizing the amounts of
intracellular free heme molecules. At the same time, the enzyme
induction could help ameliorate tissue susceptability to oxidative
stress through a reduction of excessive heme loading as well as
generation of anti-oxidant bile pigments such as bilirubin (31, 39).
With overloading of free protoheme IX, the liver can utilize several
possible mechanisms to reduce the amount of free heme molecules
overloaded in hepatocytes and to degrade them. First, once uptaken into
the parenchyma, protoheme IX could be degraded by HO-2 constitutively
expressed in the cells. Second, free protoheme IX can be captured by
empty heme-binding pockets of apo-heme proteins (e.g., cytochrome
P-450 and tryptophan 2,3-dioxygenase)
in the cells (7, 38). A portion of excessive heme molecules that
escaped from the aforementioned mechanisms could be excreted directly
into the bile (30). Finally, protoheme IX evokes transcriptional
upregulation of HO-1 in the cells and helps its oxidative degradation
through the newly induced enzyme (15). When the greater catalytic
ability of HO-1 over HO-2 (15) is considered, this mechanism could
serve as an important fail-safe mechanism that is necessary not only to
ameliorate the toxicity of protoheme IX as a prooxidant reagent but
also to help guarantee an ample perfusion in the hepatic vascular
system through the mechanism involving CO under the aforementioned
disease conditions. Considering our previous data (10, 32), these
results suggest that HO-1 and HO-2 have distinct roles in regulation of
hepatic microvascular tone under pathological and physiological
conditions, respectively.
It should be noted that CO overproduction occurred with a certain lag
time after the enzyme induction reached a maximum level. Although whole
pictures of the mechanisms are presently unknown, there are several
possible mechanisms to be considered. First, the HO reaction requires
NADPH-cytochrome P-450 reductase for an electron donor system. At 12 h, when the amounts of cytochrome P-450 become maximum, HO could share
this electron-donating system with cytochrome
P-450 monooxygenases and would not
exert its maximum catalytic activity. Second, differences in
availability of free heme molecules as a substrate of HO between naive
heme proteins and those undergoing oxidative insults should be
considered. Previous data showed that cytochrome
P-420, a denatured heme protein
derived from oxidative modification of cytochrome
P-450, serves as a better substrate
for the HO reaction than the naive protein (12). It has also been
suggested that, once exposed acutely to xenobiotics such as
acetaminophen, hepatic cytochrome
P-450 can undergo oxidative destruction through its active intermediates and can induce CO overproduction mediated by HO-2, even when the inducible enzyme is not
overexpressed (17). These previous observations led us to speculate
that availability of NADPH-mediated electron donation and oxidative
susceptability of cytochrome P-450
proteins could be factors that determine temporal discrepancy between
the HO-1 expression and CO generation. Although further investigation
is obviously necessary to solve these questions, the current results shed light on the importance of measurements of products such as CO
and/or bilirubin IX
to study the effects of HO-1 induction on organ functions.
It has been shown in the liver that at least two distinct compartments
in the vascular system are responsible for controlling the resistance:
portal veins and microvessels such as portal venules, sinusoids, and
central venules. The current study provided several lines of data
showing that, in the HO-1-induced liver, microvessels constitute a
major vascular compartment sensing the regional CO generation. First,
when administration of HbO2 or
ZnPP abolished reduction of the basal resistance and further increased
the resistance, presumably by canceling the HO-2-derived CO, there was
neither a reduction of the basal bile output nor an interlobular
heterogeneity of the perfusion, whereas a decrease in the perfusion
caused by vasoconstriction at the portal levels is known to result in
these changes (34). Second, elimination of CO by
HbO2, which can diffuse into the
space of Disse, but not by HbV, which is restricted to access the
space, preferentially constricted sinusoids in the HO-1-induced liver.
Finally, when undergoing perturbation by nanomolar levels of ET-1, a
substantial difference in the vascular resistance became evident
between the control and the HO-1-induced liver. This event can only be
explained by differences in HO-1 expression and CO generation but not
by differences of HO-2 expression between the groups, inasmuch as no
fundamental differences in the HO-2 expression in the parenchyma were
observed immunohistochemically. We were unable to observe a reduction
of the ET-1-elicited constriction of the preterminal portal venules, a
putative resistant vessel responding to low doses of this peptide
constrictor (11), in the hemin-treated liver because intravital
microscopy of the hepatic microcirculation under epi-illumination in
rats did not allow us to observe these vessels in portal regions
directly (32). However, it appears possible that vascular tone of such
portal venules can be regulated by overproduced CO, since these vessels stand in a periportal domain adjacent to hepatocytes that abundantly express HO-1 in the hemin-treated liver, as shown by
immunohistochemistry in Fig. 2. Further evaluation is obviously
necessary to specify which portion(s) of microvessels is mainly
responsible for the CO-mediated attenuation of the ET-1-induced
constriction. In summary, the current results shed light on a possible
role of CO overproduction in the mechanisms for hyporeactivity of the
hepatoportal vascular system observed in a variety of experimental
disease models accompanied by HO-1 induction, such as carbon
tetrachloride-induced cirrhosis (8), portal hypertension (9), and
hemorrhagic shock (2). Further investigation is necessary to confirm
whether the CO-mediated vasorelaxing mechanisms are applicable to
blood-perfused innervated livers in vivo under these disease models.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Takuya Tamatani and Kensuke Suzuki (Pharmaceutical
Frontier Research Laboratories, JT, Inc.) for technical support.
 |
FOOTNOTES |
This work was supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, and Culture of Japan, by grants
from Keio University School of Medicine and from Keio Medical Research
Fund, by Surveys and Research on Specific Diseases from the Ministry of
Health in 1998, and in part by Takeda Science Foundation.
R. Takamiya was an undergraduate student of Science University of
Tokyo. N. Goda is a research fellow supported by the Japan Society for
the Promotion of Science in 1998.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Ishimura,
Dept. of Biochemistry, School of Medicine, Keio Univ., 35 Shinanomachi,
Shinjuku-ku, Tokyo 160, Japan (E-mail: yishimur{at}mc.med.keio.ac.jp).
Received 21 May 1999; accepted in final form 10 August 1999.
 |
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