Extract
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Introduction
Endothelin (ET)-1 is a 21-amino-acid peptide isolated from endothelial cells. It has been found to be one of the most
potent vasoconstrictor peptides in humans and is involved in the regulation of
cerebral blood flow[1]. In addition, ET-1 has
been implicated as a mediator of cerebrovascular responses in ischemic stroke and subarachnoid hemorrhage
(SAH)[2,3]. ET-1 binds with high affinity to the endothelin-A receptor
(ETA), which mediates vasoconstriction by activating the
phospholipase C/protein kinase C cascade, decreasing smooth muscle
sensitivity to nitric oxide (NO), and increasing cytosolic free
calcium levels and superoxide anion
production[4]. The endothelin-B receptor
(ETB) has a lower affinity for ET-1 and mediates
vascular relaxation, although some vasoconstrictor
activity of ETB has also been
shown[5]. Large ET is a precursor
to endothelin with almost no vasoconstrictor activity.
ET-1 is formed when large ET-1 is cleaved by endothelin-converting
enzyme-1 (ECE-1). ECE-1 has been extensively detected in the human
brain[6], and increased serum ECE-1 activity reflects the
severity of endothelial injury to cerebral
arteries[7]. The use of ECE-1 inhibitors can prevent and reverse cerebral
vasospasm following SAH. ECE-1 inhibitors are expected to be
efficacious in the treatment of various cerebrovascular
disease[8].
TNF-α contributes to the pathology of a broad spectrum
of central nervous system diseases and injury via its action
on endothelial function. It has been shown that ET-1 can be
modulated by TNF-α. ET-1 and ECE-1 mRNA are upregulated
in response to TNF-α in endothelial
cells[9-11]. To further our understanding on how tanshinone IIA (Tan IIA) could
mediate the ET system, we examined the effect of
TNF-α on ETA and ETB mRNA, and alterations of ET-1 binding to its
receptors in the present study.
The rhizome of Salvia miltiorrhiza Bunge (SM), also
known as Tanshen, is an important herb for promoting the
circulation of blood and eliminating stasis in Chinese
traditional medicine. Previous reports have shown that SM can
prevent the postoperative increase of ET-1 after
cardiopulmonary bypass in children with congenital heart defects. It
can also inhibit ET-1 production and stimulate NO
production in human vascular endothelial and mesangial
cells[12_15]. However, the mechanisms underlying the therapeutic action
of SM are not well understood. Tan IIA is one of the major
diterpenes from SM. It can reduce brain infarct volume in
transient focal cerebral ischemia, and can markedly inhibit
the production of NO, interleukin-1β, and TNF-α, and can
suppress the expression of an inducible form of NO
synthase in activated mouse leukaemic monocyte macrophage
cell line[16]. However, the relationship between ET-1 and Tan
IIA has not been well established. As a major lipid and
soluble pharmacological constituent of SM, Tan IIA may be
involved in the interaction between SM and ET-1 in
cerebrovascular diseases. Thus, in the present study, we
exa-mined and compared the biochemical and molecular
responses of the ET system in cultured rat brain
microvascular endothelial cells (BMVEC) to TNF-α and Tan IIA in an
attempt to elucidate the possible cerebrovascular effects of
Tan IIA.
Materials and methods
Drugs and reagents RPMI-1640 medium and fetal
bovine serum (FBS) were purchased from Gibco (Grand Island,
NY, USA). TNF-α, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT), penicillin, streptomycin,
dimethylsulphoxide (Me2SO), the ET-1 immunoassay kit, and
125I-ET-1 were obtained from Sigma (St Louis, MO, USA). All
materials used were of analytical grade.
Preparation of Tan IIA extract Tan IIA was isolated from
the roots of SM, based on the method described
previously[17]. Briefly, SM was extracted with methanol at room temperature,
and then partitioned by methylene chloride, ethyl acetate, and
n-butanol in turn. The methylene chloride fraction was
subjected to column chromatography over a silica gel eluting with
a gradient system of hexane, ethyl acetate. The fractions were
combined based on their thin-layer chromatography pattern to
yield a sub-fraction designated as D1_D11. Sub-fraction D2
was further purified by repeated column chromatography over
a silica gel to afford Tan IIA. The purity of Tan IIA used was
more than 99%, which was proven by HPLC according to the
method for the assay of Tan IIA in Chinese Pharmacopoeia.
Isolation and culture of rat BMVEC Primary rat BMVEC
were isolated as previously
described[18]. Briefly, fresh rat brains were obtained from 6-week-old Wister rats, placed
into ice-cold buffer A (10 mmol/L hydroxyethyl
piperazine ethanesulfonic acid, 11.9 mmol/L
NaHCO3, 140 mmol/L NaCl, 10 mmol/L KCl, and 0.1% bovine serum
albumin). The cortices were cut into 2_3 mm pieces, further digested in a 0.1%
collagenase/dispase solution to separate the microvessels from
other components, and then centrifuged. The pellet
containing crude microvessels was further digested in a second
collagenase/dispase solution for 2 h. Microvascular
endothelial cells were purified by a Percoll gradient.
The cells were maintained in an atmosphere of 5%
CO2 at 37 °C in RPMI-1640 medium supplemented with 10% FBS,
100 U/mL penicillin, and 100 mg/mL streptomycin. The cells
of passages 3 and 4 were used for the experiments at >80%
confluency. The BMVEC were verified by staining with
factor VIII, Von Willebrand factor. The cells were stimulated
with TNF-α (5 mg/mL) in the presence or absence of Tan IIA
at the indicated concentrations. The stock solutions of Tan
IIA were dissolved in Me2SO. The concentration of
Me2SO in the final culture media was 0.1%
(v/v).
Evaluation of cell viability The cytotoxicity of Tan IIA
was evaluated via the reduced activity of MTT. The cells
were incubated for 24 h with Tan IIA at the indicated
concentrations, and some cells were activated for 8 h with
TNF-α (5 mg/mL) after the pre-incubation of Tan IIA for 24 h.
MTT (50 mg/mL) was then added to each well. The formazan
formed was dissolved in Me2SO; optical density was
measured using an ELISA microplate reader at 570 nm to represent
cellular viability. The optical density of formazan formed in
the control cells (medium alone) was taken as 100% viability.
Extraction and assay of ET-1, including large ET-1
Media samples were collected in ice-cold polypropylene tubes
containing a solution of EDTA (1 mg/mL), and aprotinin
(500 kIU/mL). The samples were extracted by addition of 1.0
mL trifluoroacetic acid (1%) in 99% distilled water and
centrifuged at 6000×g for 20 min at 4 °C. The supernatant was
loaded onto a C-18 Sep column (Waters, Milford, MA, USA)
that was previously equilibrated by washing with 100%
acetonitrile followed by 1% TFA. The peptides were eluted
slowly with 60% acetonitrile, 1% TFA, and 39% distilled water.
The eluent was collected in a clean polypropylene tube,
evaporated to dryness, and reconstituted in assay buffer. ET-1 levels,
including large ET-1, were determined using enzyme
immunoassay kits. Each kit consisted of a polystyrene 96-well
immunoplate pre-coated with a peptide antibody. Aliquots of
the reconstituted samples were loaded in duplicate onto the
wells, and the assay was carried out according to the
manufacturer's protocol. Absorbance was measured at 450
nm in an automated plate reader. The absorbance was
correlated with ET-1 concentrations, including large ET-1, to
generate a standard curve that ranged from 0 to 1000 ng/mL. The
assay of ET-1, including large ET-1, was carried out in triplicate.
Quantitation of mRNA expression by
RT-PCR Whole cell RNA was isolated by guanidine thiocyanate and cesium
chloride gradient centrifugation. Total RNA (5 mg,
determined spectrophotometrically) was used to generate
first-strand cDNA by random priming with reagents and
protocols used as recommended by the manufacturers (Pharmacia,
Freiburg, Germany; Gibco, USA). The cDNA representing
50 ng input RNA was amplified by PCR using
Taq polymerase (Gibco, USA ) in a reaction volume of 50
mL. Specific primer pairs, constructed from the reported rat gene sequence for
ET-1, ECE-1, ETA, and ETB (shown in Table 1, according to
previous reports[19_21]) were applied as described previously.
Both primer pairs were added simultaneously to the PCR
reaction vials. Each primer pair amplified a single band of the
expected size in a total volume of 50 mL for each reaction.
The following PCR profile was used: cDNA was denatured
initially for 3 min at 94 °C and then cycling started with
denaturing at 94 °C for 45 s, annealing at 54 °C for 30 s, and
extension at 72 °C for 90 s. The last cycle included a
prolonged extension at 72 °C for 7 min. For these experiments, an
optimal PCR cycling length was used for each of the primer
pairs, such that the PCR product-RNA relationship was kept
in the log-linear phase. The number of cycles chosen was 30
for the ETA and ETB receptors and ET-1, and 23 for ECE-1. All
RT-PCR experiments were routinely controlled by conducting
PCR omitting the reverse transcription. The samples were
analyzed by agarose gel electrophoresis, with the agarose gel
containing 0.4 mg/mL ethidium bromide and
0.5×Tris-acetate-EDTA buffer. The bands were visualized with 302 nm light
and photographed using a video processor (Mitsubishi,
Tokyo, Japan). Quantitative data were obtained from a
densitometer and analyzed with Quantity One 4.4.0 software
(Bio-Rad, Hercules, CA, USA). Each PCR assay was run in triplicate.
Radioligand binding studies Radiolabeled ET-1 binding
was studied in living cells. The binding medium used was
minimum essential medium with 50 mmol/L HEPES (pH 7.4),
0.1 mg/mL bacitracin, 0.1 mg/mL aprotinin, 0.48 mg/mL
leupeptin, 0.68 mg/mL pepstatin A, 0.2 mmol/L
phenyl-methylsulfonyl fluoride, and 2.5 g/L BSA. The cells
were washed 3 times with 1.0 mL binding buffer.
125I_ET-1 1×10-11
mol/L and increasing concentrations of unlabeled ET-1 in
the presence or absence of TNF-α and Tan IIA at the
indicated concentrations were added to each well. Incubations
were done at 22 °C and terminated by aspiration of the
binding medium and quick washing of the cells 3 times with
2.0 mL ice-cold binding medium. Counts were corrected for
non-specific binding or uptake by subtraction of the
radioactivity measured in the presence of excess unlabeled ligand.
Cell-bound radioactivity was counted with a Wallac 1470
gamma counter (Wallac Inc, Gaithersburg, MD, USA).
Statistical analysis Data were expressed as mean±SD.
The statistical differences among the groups were evaluated
using one-way ANOVA with Fisher's protected least
significant difference test. P<0.05 was considered significant.
Results were analyzed using SPSS 13.0 software (Spss Inc,
Chicago, TL, USA).
Results
Effects of Tan IIA on cell viability The effect of Tan IIA
on cell viability were investigated by MTT; the cells were
exposed to Tan IIA for 24 h. As shown in Figure 1, the
concentrations (0_20 mg/mL) of Tan IIA used here had no
effect on the viability of BMVEC. TNF-α at 5 mg/mL reduced
the viability of BMVEC with cell viability at 82%±3%, but
Tan IIA significantly reversed the TNF-α-induced reduction
of cell viability at concentrations of 10 and 20 mg/mL (90%±3%
and 93%±3%, respectively; P<0.05
vs 82%±3%; n=6).
Tan IIA (5 mg/mL) increased cell viability, but the difference
was not significant (85%±4%; P>0.05
vs 82%±3%; n=6).
Effects of Tan IIA on TNF-α-induced ET-1 production,
including large ET-1, in BMVEC As shown in Table
2, TNF-α significantly increased the ET-1 concentration in the media
when the BMVEC were treated with Tan IIA. TNF-α-
induced ET-1 elevation was suppressed in a
dose-dependent manner. Quite a different response pattern became
apparent for large ET-1 production. Large ET-1 levels
decreased in response to TNF-α.
Conversely, large ET-1 levels progressively increased in
response to Tan IIA in a dose-dependent manner.
Effects of Tan IIA on TNF-α-induced ECE-1 activation in
BMVEC As shown in Figure 2, the ECE-1 mRNA expression
was determined by RT-PCR. The ECE-1 mRNA expression
was significantly upregulated by TNF-α (0.97±0.13;
P<0.05 vs 0.39±0.10;
n=6). TNF-α-induced ECE-1 activation was
suppressed significantly by Tan IIA at concentrations of 10
and 20 mg/mL (0.69±0.14 and
0.58±0.06, respectively;
P<0.05 vs 0.97±0.13;
n=6). Tan IIA (5 mg/mL) downregulated
ECE-1 mRNA, but the difference was not significant
(0.84±0.09; P>0.05 vs 0.97±0.13,
n=6).
Effects of Tan IIA on TNF-α-induced mRNA expression
of ET-1 in BMVEC In order to investigate whether the
decrease in ET-1 in the media might be related to an decrease in
the ET-1 mRNA expression in BMVEC stimulated with
TNF-α, RT-PCR analysis was performed. As shown in Figure 3,
the expression of ET-1 significantly increased with
TNF-α exposure (1.13±0.13; P<0.05
vs 0.50±0.13; n=6). However, no
alteration in ET-1 mRNA could be detected after incubation
of the cells with Tan IIA at the indicated concentrations (5,
10, and 20 µg/mL) for 24 h prior to TNF-α compared with
BMVEC stimulated with TNF-α alone (1.08±0.09, 1.10±0.12,
and 1.02±0.10, respectively;
P>0.05 vs 1.13±0.13; n=6).
Effects of Tan IIA on TNF-α-induced mRNA expression
of ETA or ETB in BMVEC To evaluate the effect of Tan IIA
on the mRNA expression of ETA in BMVEC stimulated with
TNF-α, we explored the expression of
ETA or ETB with RT-PCR. As shown in Figures 4 and 5,
TNF-α exposure resulted in a significantly increased mRNA expression of
ETA (1.27±
0.09; P<0.05 vs 0.50±0.13;
n=6). However, there was no
appreciative effect on the
ETB receptor mRNA expression
(0.33±0.05; P>0.05 vs 0.34±0.04;
n=6). Compared with TNF-α alone, Tan IIA exposure resulted
in a significant decrease in the
ETA mRNA expression at concentrations of 10 and 20
mg/mL (1.00±0.12 and 0.75±0.09, respectively;
P<0.05 vs 1.27±0.09;
n=6). Tan IIA (5 mg/mL) downregulated
ETA mRNA, but the difference was not significant
(1.13±0.18; P>0.05 vs 1.27±
0.09; n=6). Tan IIA (5, 10, and 20 mg/mL) caused a significant
increase of ETB mRNA (0.42±0.04, 0.48±0.04, and 0.78±0.06,
respectively; P<0.05 vs 0.33±0.05;
n=6).
Endothelin receptor binding Receptor binding studies
were conducted to characterize the effects of Tan IIA on the
interaction of ET-1 with endothelial receptor binding. The
cells were incubated for 2 h at 37 °C with
125I-ET-1 and increasing concentrations of unlabeled ET-1 in the presence
or absence of TNF-α and Tan IIA at the indicated
concentra-tions. As shown in Figure 6, binding for
125I-ET-1 was measured. Endothelial receptor binding was unaltered in
BMVEC stimulated with TNF-α alone or with a combination
of TNF-α and Tan IIA.
Discussion
Increased circulating concentrations of TNF-α are seen
in several pathological conditions associated with vascular
disease. The effect of TNF-α was evident in endothelial
cells derived from a variety of sources. The release of ET-1
can be modulated by TNF-α[9_11].
Our findings show that TNF-α exposure led to increased levels of ET-1 and
increased ETA receptor mRNA. The increase in ET-1 secretion was
accompanied by a corresponding increase in the ET-1
gene resulting in augmented prepro ET-1 mRNA transcription
levels. Following TNF-α exposure, the ECE-1 mRNA
expression was increased, which may directly result in the
elevation of ET-1 levels with a reduction of large ET-1 levels. These
responses of BMVEC to TNF-α exposure would be
expected to exert multiple biological effects in several neurological
and pathological conditions. It has been shown that ET-1 is
produced by BMVEC in response to TNF-α to mediate
blood-brain barrier (BBB)
disruption[22]. Following both stroke and
trauma, despite the fact that TNF-α has vasodilatory action
achieved through the production of
NO[23,24], TNF-α can acutely
reduces regional cerebral blood volume, followed by
breakdown of the BBB and a reduction in
tissue water diffusion mediated via the action of ET on its
receptors[25]. TNF-α-stimulated vasoconstriction in cerebral vessels suggests that
there are regional differences in vascular sensitivity to ET-1.
The interaction between local TNF-α and ET-1 and the
resulting regional hemodynamic actions need to be further
investigated.
In pathological conditions, such as SAH and traumatic
brain injury, stimulation of the cerebral ET system seems to
play an important pathophysiological
role[1,7,26]. Accordingly, interference with the actions of ET-1 may be a worthwhile
therapeutic approach, and receptor antagonists have been
proven effective in animal experiments. However, although
preliminary clinical data has indicated the safety, there are
still limited effects of the ETA or
ETB receptor antagonists in treating cerebral vasospasm after SAH in humans. It may
provoke a drastic elevation of intracerebral ET-1
concentrations as the result of antagonists competing for ET binding
sites[27,28]. Therefore, reducing the synthesis and release of
ET-1 from endothelial or parenchymal cells may prove a
worthwhile alternative or adjunctive therapeutic option.
The results of our study, show for the first time that Tan
IIA may be involved in the processing of large ET-1 to
ET-1.This study demonstrates the inhibitory action of Tan IIA in
elevating ET-1 levels in cell culture media under stimulated
conditions. Theoretically, the decrease in ET-1 caused by
Tan IIA may be due to a decrease in production or an
increase in cellular binding/uptake or in degradation. However, an
increase in ET-1 mRNA was not detectable by RT-PCR
under these conditions. Although Tan IIA-induced decreases
in ET-1 were not accompanied by a rise in protein synthesis,
our data showed a considerable increase in the precursor
peptide of large ET-1 as well as a decrease in
ET-1. The effects of Tan IIA on ET receptor expression may lead to changes in
ET-1 levels and its biological effects. In our study, Tan IIA
exposure led to decreased
ETA receptor mRNA and increased
ETB receptor mRNA. These responses of BMVEC to Tan IIA
exposure would be expected to lead to vasodilatation of
cerebral vasculature in vivo. Moreover, an upregula-tion of
ETB receptors may lead to an increase in ET-1 binding which
will decrease ET-1 levels[29]. To determine whether the
decreased ET-1 level in BMVEC was due to enhanced
endothelial receptor binding, receptor binding studies were
conducted. We found that endothelial receptor binding was
unaltered in BMVEC stimulated with TNF-α alone or in the
combination of TNF-α and Tan IIA. Taken together, the
effect of Tan IIA may be partially attributable to a decrease in
ET-1 synthesis. The synthesis of ET-1 starts with the
generation of a prepropeptide processed by enzymes of the
constitutive secretory pathway to the immediate precursor, large
ET-1.
Large ET-1 by itself does not bind to any of the known
ET receptors; it is cleaved enzymatically, resulting in the
release of the so-called C-terminal fragment and mature ET-1.
The latter exclusively mediates the biological effects.
Enzymatic processing of large ET-1 occurs at an unusual scission
site and is the critical step in ET-1 formation. To date, 2
different specific ECE (ECE-1 and ECE-2) have been identified.
In most tissues, the expression of the ECE-1 subtype seems
to exceed that of ECE-2[30]. Accordingly, large ET-1 released
from endothelial and parenchymal cells seems to be
processed predominantly by ECE-1 activity. Therefore, our
study lends evidence to the existence of Tan IIA with
influence upon ECE-1 activity. The decreased level of ECE-1
assessed semiquantitatively in the present study confirmed
our hypothesis; it is possible that Tan IIA inhibits the
cleavage of large ET into ET-1, which would explain why Tan IIA
decreases ET-1 concentration while large ET concentrations
show a parallel increase.
The inhibition of ECE-1 by Tan IIA shown in the present
study represents a novel finding. We demonstrate for the
first time that Tan IIA decreases TNF-α-induced ET-1
expression in BMVEC through the suppression of ECE-1
synthesis. These results may at least partially explain the
cerebral vessel benefits of SM. Although suggestive,
further studies need to be carried out to elucidate the extent to
which ECE-1 is affected by Tan IIA in vitro and
in vivo, and the modulatory mechanism needs to be elucidated.
References
1 Andresen J, Shafi NI, Bryan RM. Endothelial influences on
cerebrovascular tone. J Appl Physiol 2006; 100: 318_27.
2 Yakubu MA, Shibata M, Leffler CW. Hematoma-induced
enhanced cerebral vasoconstrictions to leukotriene C4 and
endothelin-1 in piglets: role of prostanoids. Pediatric Re 1995;
38: 119_23.
3 Yakubu MA, Leffler CW. Role of endothelin-1 in cerebral
hematoma-induced modification of cerebral vascular reactivity in
piglets. Brain Res 1996; 734: 149_56.
4 Yakubu MA, Leffler CW. L-type voltage-dependent
Ca2+ channels in cerebral microvascular endothelial cells and ET-1
biosyn-thesis. Am J Physiol Cell Physiol 2002; 283: C1687_95.
5 Clozel M, Gray GA, Breu V, Loffler BM, Osterwalder R. The
endothelin ETB receptor mediates both vasodilation and
vasoconstriction in vivo. Biochem Biophys Res Commun 1995; 186:
867_73.
6 Naidoo V, Naidoo S, Mahabeer R, Raidoo DM. Cellular
distribution of the endothelin system in the human brain. J Chem
Neuroanat 2004; 27: 87_98.
7 Juvela S. Plasma endothelin and big endothelin concentrations
and serum endothelin-converting enzyme activity following
aneurysmal subarachnoid hemorrhage. J Neurosurg 2002; 97:1287_93.
8 Kwan AL, Lin CL, Chang CZ, Wu SC, Howng SL, Jeng AY.
Attenuation of SAH-induced cerebral vasospasm by a selective ECE
inhibitor. Neuroreport 2002; 13: 197_9.
9 Marsden PA, Brenner BM. Transcriptional regulation of the
endothelin-1 gene by TNF-αlpha. J Physiol Cell Physiol 1992;
262: 854_61.
10 Zhao RZ, Chen X, Yao Q, Chen C. TNF-αlpha induces
interleukin-8 and endothelin-1 expression in human endothelial cells with
different redox pathways. Biochem Biophys Res Commun 2005;
327: 985_92.
11 Molet S, Furukawa K, Maghazechi A, Hamid Q, Giaid A.
Chemokine- and cytokine-induced expression of endothelin-1
and endothelin-converting enzyme-1 in endothelial cells. J
Allergy Clin Immunol 2000; 105: 333_8.
12 Ji XY, Tan BKH, Zhu YZ. Salvia
miltiorrhiza and ischemic diseases. Acta Pharmacol Sin 2000; 21: 1089_94.
13 Chan K, Chui SH, Wong DY, Ha WY, Chan CL, Wong RN.
Protective effects of Danshensu from the aqueous extract of
Salvia miltiorrhiza (Danshen) against homocysteine-induced
endothelial dysfunction. Life Sci 2004; 75: 3157_71.
14 Xu M, Wang YP, Luo WB, Xuan LJ. Salvianolate inhibits
proliferation and endothelin release in cultured rat mesangial cells.
Acta Pharmacol Sin 2001; 22: 629_33.
15 Xia ZY, Gu JZ, Ansley DM, Xia F, Yu JF. Antioxidant therapy
with Salvia miltiorrhiza decreases plasma endothelin-1 and
thromboxane B2 after cardiopulmonary bypass in patients with
congenital heart disease. J Thorac Cardiovasc Surg 2003; 126:
1404_10.
16 Jang SI, Jeong SI, Kim KJ, Kim HJ, Yu HH, Park R,
et al. Tanshinone IIA from Salvia
miltiorrhiza inhibits expression of inducible
nitric oxide synthase and production of TNF-α,
IL-1β and IL-6 in activated RAW 264.7 cells. Planta Med 2003; 69: 1057_9
17 Kim HH, Kim JH, Kwak HB, Huang H, Han SH, Ha
H, et al. Inhibition of osteoclast differentiation and bone resorption by
tanshinone IIA isolated from Salvia
miltiorrhiza. Bunge Biochem Pharmacol 2004; 67: 1647_56.
18 Abbott NJ, Hughes CC, Revest PA, Greenwood J. Development
and characterisation of a rat brain capillary endothelial culture:
towards an in vitro blood-brain barrier. J Cell Sci 1992; 103:
23_37.
19 Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Coto K, Masaki
T. Cloning of a cDNA encoding a non-isopeptide-selective
subtype of the endothelia receptor. Nature 1990; 348: 732_5.
20 Sakurai T, Yanagisawa M, Inoue A, Ryan US, Kimura S, Mitsui Y,
et al. cDNA cloning, sequence analysis and tissue distribution of
rat preproendothelin-1 mRNA. Biochem Biophys Res Commun
1991; 175: 44_7.
21 Shimada K, Takahashi M, Tanzawa K. Cloning and functional
expression of endothelial-converting enzyme from rat BMVEC.
J Biol Chem 1994; 269: 18 275_8.
22 Didier N, Romero IA, Creminon C, Wijkhuisen A, Grassi J,
Mabondzo A. Secretion of interleukin-1β by astrocytes mediates
endothelin-1 and tumour necrosis factor-α effects on human
brain microvascular endothelial cell permeability. J Neurochem
2003; 86: 246_54.
23 Macnaul KL, Hutchinson NI. Differential expression of iNOS
and cNOS mRNA in human vascular smooth muscle cells and
endothelial cells under normal and inflammatory conditions.
Biochem Biophys Res Commun 1993; 196: 1330_4.
24 Hollenberg SM, Cunnion RE, Parrillo JE. The effect of tumor
necrosis factor on vascular smooth muscle. In
vitro studies using rat aortic rings. Chest 1991; 100: 1133_7.
25 Sibson NR, Blamire AM, Perry VH, Gauldie J, Styles P, Anthony
DC. TNF-αlpha reduces cerebral blood volume and disrupts tissue
homeostasis via an endothelin- and NFR2-dependent pathway.
Brain 2002; 125: 2446_59.
26 Armstead WM. Endothelin-induced cyclooxygenase-dependent
superoxide generation contributes to K+ channel functional
impairment after brain injury. J Neurotrauma 2001; 18: 1039_48.
27 Plumpton C, Ferro CJ, Haynes WG, Webb DJ, Davenport AP.
The increase in human plasma immunoreactive endothelin but
not big endothelin-1 or its C-terminal fragment induced by
systemic administration of the endothelin antagonist TAK-044. Br
J Pharmacol 1996; 119: 311_4.
28 Chuquet J, Benchenane K, Toutain J, MacKenzie ET, Roussel S,
Touzani O. Selective blockade of endothelin-B receptors
exacerbates ischemic brain damage in the rat.
Stroke 2002; 33: 3019_24.
29 Pollock DM, Allcock GH, Krishnan A, Dayton BD, Pollock JS.
Upregulation of endothelin B receptors in kidneys of DOCA-salt
hypertensive rats. Am J Physiol Renal Physiol 2000; 278:
279_86.
30 Parnot C, Le Moullec JM, Cousin MA, Guedin D, Corvol P, Pinet
F. A live-cell assay for studying extracellular and intracellular
endothelin-converting enzyme activity. Hypertension 1997;
31: 837_44.
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