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Introduction
Pulmonary hypertension (PH) is a common complication of congenital heart defects, with a left-to-right shunt
characterized by high pulmonary blood flow. Pulmonary vascular structural remodeling (PVSR) is the pathological basis of PH. To
date, the pathophysiological features and mechanisms responsible for PH and PVSR induced by increased pulmonary blood
flow have not been fully understood. Some studies demonstrated that high pulmonary blood flow could produce high shear
stress on pulmonary artery endothelial cells (EC) and smooth muscle cells (SMC), resulting in a maladjustment of balance in
maintaining the structure and function of the vascular wall. Nitric oxide (NO) and carbon monoxide (CO) were found to play
an important regulatory role in the development of PH and
PVSR[1_3].
Hydrogen sulfide (H2S), a well-known toxic gas with the smell of rotten eggs whose toxicity was first described in 1713,
was proven to be generated in vivo in human and animal organisms and participates in many pathophysiological processes.
H2S, as a novel gaseous messenger molecule, is generated endogenously in mammalian tissues from
L-cysteine metabolism mainly by 3 enzymes: cystathionine
b-synthetase (CBS), cystathionine g-lyase (CSE) and 3-mercaptosulfurtransferase
(MST)[4_6]. The distribution of these 3 enzymes is tissue specific, and CSE seems to be the only rate-limiting enzyme to catalyze
H2S metabolism in the cardiovascular
system[4]. Previous studies by our laboratory and other researchers showed that
H2S played a regulatory role in pathophysiological processes in the
cardiovascular[7_12], nervous[13]
and other systems[14,15]. However, the potential significance of
H2S in the pathogenesis of PH and PVSR was unclear. NO is a vasorelaxant factor
produced mainly in vascular endothelial cells, while CO and
H2S are smooth muscle cell-derived factors. As small gas
molecules, they are characterized by common features such as low molecular weight, continuous release and quick dispersal
and absorbance, which makes it possible for them to interact. Previous studies, including ours, indicated the possible
interaction between NO and
H2S[8,16,17] and CO and
H2S[18,19] under physiological and pathological conditions. Therefore, the
present study was designed to determine whether
H2S was involved in the development of PH and PVSR and whether
H2S had an impact on the NO/NO synthase (NOS) pathway and CO/heme oxygenase (HO) pathway in a rat model of PH and PVSR
induced by high pulmonary flow.
Materials and methods
Animal model of pulmonary hypertension The experiment was conducted in accordance with the Guide for the Care and
Use of Experimental Animals issued by the Ministry of Health of the People¡¯s Republic of China. Male Sprague-Dawley rats
were provided by the Animal Research Centre of Peking University First Hospital. The rats were housed in plastic cages in
a room with a controlled humidity of 40%, at temperature of 22
°C and 12 h light-cycle from 06:00 to 18:00. The animal model
was established by an abdominal aorta-inferior vena cava shunt operation according to the method described by Garcia and
Diebold[20], with some
modifications[21]. Thirty two rats, weighing 120_140 g, were randomly divided into the following
groups: shunt (n=8), shunt+NaHS (sodium hydrosulfide)
(n=8), sham (n=8) and sham+NaHS
(n=8). Rats in the shunt and shunt+NaHS groups were anesthetized with 0.25% pentobarbital sodium (40 mg/kg, intraperitoneal injection). The
abdominal aorta and inferior vena cava were exposed, and then a bulldog vascular clamp was placed across the aorta cauda to the left
renal artery. The aorta was punctured at the union of the segment two-thirds caudal to the renal artery and one-third cephalic
to the aortic bifurcation with use of an 18-gauge disposable needle. The needle was then slowly withdrawn and a 9_0 silk
thread was used to stitch the puncture of the abdominal wall. Rats in the sham and sham+NaHS groups underwent the same
experimental protocol as mentioned above except for the shunt procedure. Rats in the shunt+NaHS and sham+NaHS groups
were intraperitoneally injected with NaHS at 56 mmol/kg each day for 11 weeks as
described[7]. Rats in the shunt and sham
groups were injected with the same volume of physiological saline.
Blood gas analysis and measurement of
Qp/Qs Blood samples (0.5 mL) were obtained from the pulmonary artery, external
carotid artery and jugular vein for blood gas analysis with use of a Blood Gas Analysis Apparatus (GASTAT-3, Japan).
Qp/Qs was calculated by the formula:
Qp/Qs=[oxygen saturation of aorta (%)-oxygen saturation of inferior vena cava (%)]/[oxygen
saturation of pulmonary vein (%)-oxygen saturation of pulmonary artery (%)]. When the oxygen saturation of the aorta was
>95%, the oxygen saturation of the pulmonary vein was considered 100%. When the oxygen saturation of the aorta was
<95%, the oxygen saturation of pulmonary vein was considered 95%. The ratio
Qp/Qs was calculated as an indicator to
evaluate the pulmonary flow and body flow.
Measurement of hemodynamic parameters and sample
preparation At the end of the experiment, the animals were
weighed and anesthetized with pentobarbital sodium (40 mg/kg,
intraperitoneal injection). A silicone catheter (0.9 mm in outer
diameter) was induced under fluoroscope into the right jugular vein via a venotomy and passed across the tricuspid valve
and right ventricle into the pulmonary artery. The other end of the catheter was connected to a transducer (YZ-05-1, Beijing,
China). The systolic pulmonary artery pressure (SPAP) was simultaneously recorded. Then the heart was removed and the
right ventricle (RV) and the left ventricle (LV) plus septum (SP) were dissected. These tissues were weighed on an electronic
scale. The ratio of wet weight of right ventricle to left ventricle plus SP [RV/(LV+SP)] was calculated as an indicator of right
ventricular hypertrophy. The right side of the lung tissue was removed and kept in liquid nitrogen for quick freezing and then
stored at -80 ºC for homogenate. The left lower part of lung tissue was removed to 10% (w/v) paraformaldehyde for fixation.
A small part of lung tissue was cut from the upper part of the left lung lobe and quickly immersed into 3% glutaraldehyde.
Morphological analysis of small pulmonary arteries
Lung tissue fixed in 10% paraformaldehyde was dehydrated,
embedded in paraffin, and cut in sections 4-µm thick. The elastic fiber in lung tissues was stained according to the modified
Weigert¡¯s method and counterstained with Van Gieson solution. Morphological analysis was by use of a video-linked
microscope digitizing board system (Leica Q550CW, Germany). Only vessels showing clearly defined external and internal
elastic lamina were analyzed. In accordance with Bath
et al[22], the relative medial thickness (RMT) and relative medial areas
(RMA) of intra-acinar pulmonary arteries were calculated. The ratio of muscular artery (MA), partially muscular artery (PMA)
and non-muscular artery (NMA) were also observed. Lung tissue fixed in 3% glutaraldehyde was cut into 1 mm×1 mm×1 mm
pieces and post-fixed in 1% phosphate-buffered osmium tetroxide for 6 h, then rinsed again (overnight) and dehydrated in a
graded series of ethanol. These specimens were infiltrated with propylene oxide and embedded with Epon 812. These
procedures were all done at room temperature. Finally, specimens were embedded in new batches of Epon 812 and
polymerized at 40 ºC (24 h) and 60 ºC (48 h). Series of transverse or longitudinal sections were obtained from blocks. Semi-thin
sections (1 µm) were stained with azur-II and methylene blue. Ultra-thin sections (60_90 nm) were made with use of an ultra
microtome and mounted on formvar-coated copper grids (75 meshes), then stained with uranylacetate and lead citrate and
examined under a transmission electron microscope (JEM-100CX, JEOL, Japan) in detail.
Western blotting Lung tissues were lysed in a lysis buffer (1 mol/L Tris-Cl 0.2 mL, pH 8.0, 5 mol/L NaCl 0.3 mL, 500
mmol/L edetic acid 10 µL, 100 mmol/L PMSF 0.1 mL, Triton X-100 10 µL, adding water to 10 mL). The extracts were clarified by
centrifugation at 12 000 rpm for 15 min at 4 ºC. SDS-PAGE and Western blot analysis was performed according to experimental
protocol. The primary antibody dilutions were as follows: 1:200 for eNOS (Santa Cruz Biotechno-logy, Santa Cruz, CA, USA),
1:100 for HO-1 (Sigma, St Louis, MO, USA), 1:200 for extracellular signal-regulated kinase (ERK) (Santa Cruz Biotechnology),
1:200 for phosphorylation ERK (P-ERK) (Santa Cruz Biotechnology), 1:200 for proliferation cell nuclear antigen (PCNA)
(Santa Cruz Biotechnology) and 1:5000 for b-actin (TBD, USA). HRP-conjugated secondary antibody was used at 1:5000.
The immunoreactions were visualized by ECL and exposed to
X-ray film (Kodak Scientific Imaging film, X-omat Blue XB-1, USA).
Assay of H2S in lung tissue Lung tissue was homogenized in a 10-fold volume (w/v) of an ice-cold potassium phosphate
buffer (pH 6.8). The reaction was performed in 25 mL in an Erlenmeyer Pyrex flask. Cryovial test tubes (2 mL) were used as
the center wells, each containing 1 mol/L NaOH of 0.5 mL. The reaction mixture contained lung tissue homogenate and 1
mol/L HCl at a ratio of 1:5. The flasks containing reaction mixture and central wells were flushed with
N2 30 s before being sealed with a double layer of parafilm. The reaction was initiated by transferring the flasks from ice to a shaking water bath at 37
°C. After incubation at 37 °C
for 4 h, the contents of the central wells were then transferred to 10-mL beakers, each containing 0.5 mL
of antioxidant solution. Subsequently, the solution was measured with sulfide-sensitive electrodes (PXS270, Shanghai,
China), and the lung tissue H2S content was calculated against the calibration curve of the standard
H2S solution.
Measurement of NO and NOS activity in lung tissue
In accordance with the methods described by Stathopulos
et al[23], with modification, NO production and NOS activity in lung tissue were measured at 540 nm with use of a Spectrophotometer
(DU 6400; Beckman).
Measurement of CO in lung tissue CO content in lung tissue was detected according to the methods described by
Morita and Kourembanas[24] and
Chalmer[25], with modifica-tion. The lung tissue homogenate was centrifuged at 12 000 rpm
for 20 min and 0.5 mL of the supernatant was mixed with 1 mL of the hemoglobin solution, which was derived from a mixture
of 0.25 mL of fresh packed erythrocytes from rats and 50 mL of 0.25 mol/L ammonia solution. Then 0.1 mL of sodium dithionite
was added. The absorbance of the tested lung tissue homogenate sample and water blank at 568 nm and 581 nm was read and
the ratio (R) of absorbance at the 568 and 581 nm readings was calculated. Then the percentage of HbCO from a standard
curve derived by mixing different proportions of 2 Hb solutions containing 100% HbCO and 100%
HbO2 was derived. Lung tissue homogenate CO was then calculated as follows: CO (nmol/µg)=% HbCO×Hb (mg/L)×4×4×1000/(64 456×100×X). X
represents the volume of homogenate.
Statistical analysis Data were expressed as mean±SD and statistically analyzed by use of SPSS 10.0. Comparison among
groups involved one-way ANOVA followed by Student-Neuman-Keuls¡¯ tests. A value of
P<0.05 was considered statistically significant.
Results
Changes in
Qp/Qs and hemodynamic
parameters In the present rat model of abdominal aorta-inferior vena cava shunt,
Qp/Qs in the shunt-alone group and shunt+NaHS group increased significantly as compared with that of sham group
(P<0.01). The shunt and shunt+NaHS groups did not differ significantly in
Qp/Qs. As shown in Table 1, the SPAP increased
significantly in rats of the shunt-alone group as compared with that of the sham group
(P<0.01), whereas the SPAP in rats of the
shunt+NaHS group decreased significantly as compared with that of the shunt-alone group
(P<0.01). The SPAP between the sham and sham+NaHS
groups, however, did not differ significantly. RV/(LV+SP) was higher in rats of shunt-alone group than that of the sham
group (P<0.01). Whereas RV/(LV+SP) was lower in rats of the shunt+ NaHS group than that of shunt-alone group
(P<0.01). The sham and sham+NaHS groups, however, did not differ significantly
in RV/(LV+SP) (Table 1).
Administration of exogenous H2S donor alleviated
pulmonary vascular structural remodeling As shown in Table 2, the
proportion of MA and PMA increased significantly, whereas NMA decreased significantly in the rats of the shunt-alone
group as compared with that of sham group (all
P<
0.01). After the administration of exogenous
H2S donor for 11 weeks, the proportion of MA and PMA decreased significantly,
and that of NMA increased as compared with the shunt-alone group (all
P<0.01). The sham and sham+ NaHS groups did not
differ significantly in proportion to MA, PMA, and NMA.
RMT and RMA of intra-acinar pulmonary arteries increased significantly in rats of shunt-alone group as compared with
that of the sham group (all P<0.01). After administration of NaHS for 11 weeks, RMT and RMA decreased significantly as
compared with shunt-alone group (all P<
0.01). The sham and sham+NaHS groups did not differ significantly in RMT and RMA proportions (Figure 1).
After 11 weeks of shunting, the ultra-structure of the intra-acinar pulmonary arteries showed swollen and hypertrophic
endothelial cells, irregular internal elastic lamina and increased rough endoplasmic reticulum and free ribosomes in the
cytoplasm of SMC. After administration of NaHS for 11 weeks, the intra-acinar pulmonary arteries showed the flat endothelial
cells, regular internal elastic lamina and cytoplasm of SMC full of myofilaments and dense bodies (Figure 2).
Administration of exogenous H2S donor impacted pulmonary vascular structural remodeling and ERK/MAPK
pathway PCNA expression of the pulmonary artery increased significantly in rats of the shunt-alone group as compared with that of
the sham group (P<0.01). After administration of NaHS for 11 weeks, compared with that of the shunt-alone group, the
shunt+NaHS group showed significantly decreased PCNA expression in pulmonary artery
(P<0.01). The sham and sham+NaHS groups did not differ significantly in PCNA expression of the pulmonary artery (Figure 3).
The ratio of P-ERK/ERK1 expression of pulmonary arteries increased significantly in rats of the shunt-alone group as
compared with that of the sham group (P<0.01). After administration of NaHS for 11 weeks, the ratio of P-ERK/ERK1
expression of pulmonary arteries decreased significantly as compared with the shunt-alone group
(P<0.01). The sham and sham+NaHS groups did not differ significantly in the ratio of P-ERK/ERK1 expression of the pulmonary artery (Figure 4).
Administration of exogenous H2S donor increased the level of lung tissue
H2S The production of lung tissue
H2S in rats of the shunt-alone group was lower than that of the sham group. After administration of NaHS for 11 weeks in rats of the
shunt+NaHS and sham+NaHS groups, the level of
H2S increased significantly (P<0.01) (Table 3).
Administration of exogenous H2S donor attenuated the increased production of endogenous NO, NOS activity and eNOS
protein in lung tissue As shown in Table 3, the production of endogenous NO increased significantly in rats of the
shunt-alone group as compared with those of the sham group
(P<0.01). After treatment with NaHS for 11 weeks, the production of
endogenous NO decreased significantly compared with the shunt-alone group
(P<0.01). The sham and sham+NaHS groups
did not differ significantly in the production of endogenous NO. Corresponding to the changes in the production of
endogenous lung tissue NO, NOS activity increased significantly in rats of the shunt-alone group as compared with that of
the sham group (P<0.01). In contrast to the shunt-alone group, the shunt+NaHS group showed that NOS activity decreased
by 95.11% in lung tissue (P<0.01). The sham and sham+NaHS groups did not differ significantly in NOS activity in lung
tissue (Table 3).
Lung tissue eNOS protein expression in rats of the shunt-alone group was up-regulated significantly compared with that
of the sham group (P<0.01). In contrast to the shunt-alone group, the shunt+NaHS group showed significantly
down-regulated eNOS protein expression in lung tissue
(P<0.01). The sham and sham+NaHS groups did not differ significantly in eNOS protein expression in lung tissue (Figure 5).
Administration of exogenous H2S donor increased the production of endogenous CO and expression of HO-1 protein in
lung tissue The production of endogenous CO between the sham and shunt-alone groups did not significantly differ. In
contrast to the shunt-alone group, the shunt+NaHS group showed a significant increase in the production of endogenous
CO (P<0.01). The sham and sham+NaHS groups did not differ significantly in the production of endogenous CO (Table 3).
The changes in lung tissue HO-1 protein expression were consistent with those of the production of endogenous CO.
The lung tissue HO-1 protein expression between the sham and shunt-alone groups did not differ significantly. Lung tissue
HO-1 protein expression increased significantly in rats of the shunt+NaHS group as compared with that of the shunt-alone
group (P<0.01). The sham and sham+NaHS groups did not differ significantly in the lung tissue HO-1 protein expression
(Figure 6).
Discussion
In the present study, we successfully established the rat model of aortocaval shunt. The mortality was 5.9% of the 17 rats
with an aortocaval shunt one died within 24 h of shunt implantation because of a congested lung as was seen on autopsy.
After 11 weeks of shunting, the SPAP increased by
48.63%, which suggested that shunt rats with high pulmonary blood flow developed PH. Furthermore, the proportion of MA
and PMA increased by 74.20% and 90.99%, respectively, whereas that of NMA decreased by 32.17%, and the RMT and RMA
of the intra-acinar pulmonary arteries increased by 86.92% and 39.91%, respectively. The ultrastructure of the intra-acinar
pulmonary arteries showed swollen and hypertrophic endothelial cells, irregular internal elastic lamina and increased rough
endoplasmic reticulum and free ribosomes in the cytoplasm of SMC. The above findings demonstrated that PVSR was
characterized by increased mascularization and ultrastructural changes of small pulmonary arteries, along with the
development of PH.
The mechanisms responsible for PH and PVSR induced by high pulmonary flow have not been fully understood. Studies
showed that endothelial cells of pulmonary arteries were constantly exposed to fluid mechanical forces derived by
high-flowing blood. These forces, especially shear stress, caused progression from endothelial dysfunction to smooth muscle
dysfunction as vascular changes progress, resulting in the PH and
PVSR[26]. Many cytokines and growth factors, such as
basic fibroblast growth factor, transforming growth factor
b, epidermal growth factor, platelet derived growth factor and
endothelin, are implicated as contributing
fact-ors[26_28]. However, the mechanism for PVSR has not been understood. In the
1980s, NO and CO were determined to be gaseous messenger molecules sharing common features such as low molecular
weight, continuous release and quick dispersal and absorbance. The discovery that NO has powerful vasoactive properties
identical to those of endothelial-derived relaxing factor spawned a vast body of research investigating the physiological
actions of small gas molecules in PH and
PVSR[29]. Like NO, CO acts as a vasorelaxant and regulates other vascular functions
such as smooth muscle proliferation. CO, when applied at low concentrations, exerts potent cytoprotective effects mimicking
those of HO-1 induction in PH and
PVSR[30]. However, many questions are still unanswered regarding the mechanisms
responsible for the development of PH and PVSR.
H2S, the newly discovered third endogenous gasotrans-mitter, was known as a toxic gas for hundreds of years, with little
attention paid to its physiological and pathophysiological function. Studies have demonstrated the biological functions of
H2S such as hyperpolarization of cell
membranes[4], relaxation of
SMC[4], inhibition of SMC
proliferation[31], and decrease in neuronal
excitability[32]. Our recent studies showed that
H2S in some cardiovascular diseases played a regulatory role, such
as myocardial injury induced by
isoproterenol[12], hypertension induced by inhibition of
NOS[8], spontaneous
hypertension[7] and hypoxic pulmonary
hypertension[9]. However, whether and how
H2S plays a role in PH and PVSR induced by high
pulmonary blood flow is still unknown.
To seek the answer to this question, we measured the production of lung tissue
H2S in shunted rats. After 11 weeks of
shunting, PH, and PVSR resulted from high pulmonary blood flow. Interestingly, the production of lung tissue
H2S in shunted rats was decreased by 33.09%.
To further clarify the possible role of
H2S in PH and PVSR induced by high pulmonary blood flow, NaHS, a donor of
exogenous H2S was supplied for 11 weeks, and it significantly increased the production of lung tissue
H2S. At the same time, SPAP decreased by 19.82% and RV/(LV+SP) decreased by 7.3%, which suggested that the increased
H2S could decrease pulmonary pressure and attenuate right ventricular hypertrophy in rats with high pulmonary flow. Also, after administration
of NaHS for 11 weeks, the proportion of MA and PMA decreased by 14.37% and 12.18%, respectively, and that of NMA
increased by 13.96%. RMT and RMA decreased by 14.26% and 14.33%, respectively. The intra-acinar pulmonary arteries
showed flat endothelial cells, regular internal elastic lamina and a cytoplasm of SMC full of myofilaments and dense bodies.
The findings above demonstrated that NaHS alleviated PVSR induced by high pulmonary blood flow in rats.
However, the mechanisms by which H2S regulates PH and PVSR are not fully understood. Vascular smooth muscle cell
(VSMC) proliferation resulted in vascular wall thickening and increased pulmonary vascular resistance, which is the
pathophysiological base of PH and PVSR. Our previous
in vitro study showed that H2S could inhibit VSMC proliferation in the
cultured cells of rats[27]. Yang et
al reported that CSE overexpression could inhibit cell proliferation
in vitro[33]. However, whether
H2S affects VSMC proliferation and its roles
in vivo have not been reported. In this experiment, we found that PVSR
was characterized by thickened medial membrane in shunted rats, and administration of NaHS for 11 weeks alleviated the
thickening, as shown by thinned medial membrane in shunted rats. To explore whether PVSR is involved in VSMC proliferation,
PCNA expression was investigated. After 11 weeks of shunting, PCNA protein expression of the pulmonary artery increased
significantly. After administration of NaHS for 11 weeks, PCNA protein expression decreased significantly in pulmonary
arteries. The results suggest that exogenous
H2S attenuated the increased pulmonary SMC proliferation in rats with high
pulmonary blood flow in vivo.
The MAPK cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and
differentiation. MAPK-catalyzed phosphorylation of substrate proteins functions as a switch to turn on or off the activity of
the substrate protein. The ERK pathway has been shown to be involved in the stimulation of cellular
proliferation[34]. The ratio of P-ERK/ERK indicates the degree of activation of the ERK/MAPK pathway. In this experiment, after 11 weeks of
shunting, the ratio of P-ERK/ERK1 in pulmonary arteries increased significantly. After the administration of NaHS for 11
weeks, the ratio of P-ERK/ERK1 in pulmonary arteries decreased significantly. Nevertheless, whether
H2S inhibited VSMC proliferation by the ERK/MAPK pathway in rats with high pulmonary blood flow
in vivo needs to be studied further.
NO and CO are both important gasotransmitters formed in vessels. They play a part in the regulation of PVSR. Our
previous study showed a possible interaction between
H2S and NO and between H2S and CO in blood
vessels[17,18,35]. However, little is known about the impact of
H2S on the NO/NOS and CO/HO pathways in a rat model of PH and PVSR
induced by high pulmonary blood flow. To further understand the possible mechanism by which
H2S regulated PH and PVSR in rats with high pulmonary blood flow, we investigated the lung tissue NO, NOS activity and eNOS protein. It was shown
that, after 11 weeks of shunting, the production of endogenous NO, NOS activity and eNOS protein expression increased
significantly. After treatment with NaHS for 11 weeks, the production of endogenous NO, NOS activity and eNOS protein
expression decreased significantly. The above findings showed that exogenous
H2S attenuated the activated NO/NOS pathway in shunted rats, which might be involved in the mechanism by which
H2S alleviates PVSR.
CO, another gaseous small molecule, has been reported to inhibit VSMC proliferation in hypoxic pulmonary
hypertension[3]. In the present rat model, we found that, after treatment with NaHS for 11 weeks, the production of endogenous CO
increased significantly and HO-1 protein expression in lung tissue was up-regulated significantly.
How H2S affects the NO/NOS and CO/HO pathways is still unknown in the pathogenesis of PH induced by high
pulmonary blood flow. NO, CO, and H2S were considered as gasotransmitters, therefore we postulated that NO, CO, and
H2S might possibly interact under physiological and pathological conditions, and they might constitute a regulatory network in the
vascular system. In the present study, NaHS increased the level of
H2S but decreased the generation of lung tissue NO, NOS
activity and eNOS protein expression, which suggested that
H2S might inhibit the generation of endogenous NO through
down-regulation of eNOS protein expression and decreased NOS activity. However, whether changes in the NO pathway are
involved in NaHS regulating PVSR needs to be studied further.
NaHS increased the level of lung tissue
H2S but facilitated the generation of CO, by up-regulating HO-1 protein expression.
H2S can be scavenged by methemoglobin, or metallo- or disulfide-containing molecules such as oxidized
glutathione[36]. Hemoglobin may be the common "sink" for CO in forming
carboxyhemoglobin[37], and for
H2S in forming green sulhemoglobin. We speculate that
H2S might directly affect carboxyhemoglobin metabolism resulting in the release of CO under pathological
conditions. Nevertheless, the molecular mechanism by which
H2S regulates the CO/HO pathway needs to be studied further.
H2S also has a direct effect on the vasculature, which might be involved in the mechanisms by which
H2S attenuated PH. Previous studies showed that
H2S exerted a direct effect on vessels as a vasodilator
in vitro[4,16], and its mechanism was
related to the opening of the KATP channel .
NaHS was used as a donor of H2S in this study for the following reasons:
(1) It was reported that about one-third of
H2S existed as the undissociated form
(H2S) and the remaining two-thirds as
HS_ at equilibrium with H2S in physiological
saline[27]. NaHS dissociates to
Na+ and HS- in solution and then
HS- associates with H+ and produces
H2S. (2) A bolus intraperitoneal injection of NaHS leading to an increase in plasma
H2S level and a decrease in mean arterial blood pressure continued at least
for 6 h in rats[8], much longer than a transient decrease in blood pressure (lasting minutes) caused by an intravenous bolus
injection of gaseous H2S [4], and the use of NaHS enables us to define the concentrations of
H2S in solution more accurately and reproducibly than bubbling
H2S gas. (3) NaHS at concentrations used in the present study does not change the pH of
the medium.
In conclusion, this study provided evidence for the first time that
H2S exerted a regulatory effect on PH and PVSR induced
by high pulmonary blood flow. Meanwhile,
H2S down-regulated the ERK/MAPK signal pathway, inhibited the NO/NOS
pathway and enhanced the CO/HO pathway in rats with high pulmonary blood flow. Whether
H2S could be a therapeutic target for PH and PVSR induced by high pulmonary blood flow needs further exploration.
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