718
Release of Immunoreactive Endothelin From
Porcine Aortic Strips
Masakazu Kohno, Kenichi Yasunari, Koh-ichi Murakawa, Koji Yokokawa, Takeshi Horio,
Toshiki Fukui, and Tadanao Takeda
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The present study was designed to determine whether endothelin and "big endothelin" are
released from aortic strips with endothelium and to examine the effect of thrombin by using a
specific radioimmunoassay. Porcine aortic strips with endothelium released immunoreactive
endothelin (ir-endothelin) and immunoreactive big endothelin (ir-big endothelin) into the
medium in a time-dependent manner. These releases were markedly inhibited by 10 fig/ml
cycloheximide. Expectedly, after removal of endothelium, aortic strips did not release a
detectable amount of ir-endothelin and ir-big endothelin. In contrast, thrombin (10 units/ml)
significantly stimulated the release of ir-endothelin and ir-big endothelin. Reverse-phase
high-performance liquid chromatography coupled with radioimmunoassay revealed that the
major component of ir-endothelin corresponds to standard endothelin-1 (1-21) and the major
component of ir-big endothelin corresponds to standard big endothelin (porcine, 1-39). These
results suggest that aortic strips with endothelium release endothelin and big endothelin slowly
but continuously into the extracellular space and that these releases can be stimulated by
thrombin. (Hypertension 1990;15:718-723)
T
he role of the vascular endothelium in the
regulation of vascular smooth muscle tonus
has been the focus of much interest in recent
years. Previous studies have described a proteasesensitive vasoconstrictor activity in supernatants of
cultured endothelial cells (ECs).1-3 Recently, Yanagisawa et al4 isolated an endothelium-derived 21residue vasoconstrictive peptide, endothelin, from
the culture supernatant of porcine aortic ECs, determined its amino acid sequence, and molecularly
cloned the peptide precursor. This peptide has a
potent and long-lasting vasoconstrictor activity and is
closely associated with Ca2+ influx probably through
dihydropyridine-sensitive Ca2+ channels.4-5 We have
recently shown that the intravenous infusion of synthetic endothelin-1 causes a characteristically longlasting elevation in blood pressure and a pronounced
reduction in renal blood flow in rats.6 Furthermore,
endothelin is known to cause a lasting elevation in
intracellular Ca2+ in cultured vascular smooth muscle
cells.7-9 These observations strongly suggest that
endothelin plays a role in the regulation of vascular
tonus.
From the First Department of Internal Medicine, Osaka City
University Medical School, Osaka, Japan.
Supported by Grant-in-Aid for Scientific Research 6148210
from the Ministry of Education, Science, and Culture, Japan.
Address for correspondence: Masakazu Kohno, MD, The First
Department of Internal Medicine, Osaka City University Medical
School, 1-5-7, Asahi-machi, Abeno-ku, Osaka 545, Japan.
In contrast, we have observed that cultured ECs
isolated from porcine aortas release immunoreactive
endothelin (ir-endothelin) into the medium in a
time-dependent manner.10 This finding, however,
does not necessarily mean that produced endothelin
is released from aortic ECs in vivo and acts as a
circulating hormone because cultured ECs can more
closely approximate a state of "injury."11 Therefore,
the present experiment was designed to determine
whether endothelin is released from aortic strips with
endothelium, not cultured ECs, into the extracellular
space and to examine the effect of thrombin on this
release by using a radioimmunoassay.10'12 Moreover,
we also examined whether "big endothelin," an
intermediate form with 39 amino acid residues, is
released from aortic strips with endothelium.
Methods
Preparation of Aortic Strips
The experiments were performed on strips of adult
porcine thoracic aortas. Thoracic aortas were immediately removed and suspended in cold serum-free
Dulbecco's modified Eagle's medium (DMEM) that
contained penicillin (100 fig/m\) and streptomycin
(50 jig/ml) and then were transported to our laboratory. All side branches and their connective tissue
were then removed. This aortic segment was cut
transversely for a length of 20 cm and vertically for a
width of 4 cm. This aortic segment (4x20 cm) was
gently cut into 20 pieces of 2x2-cm strips. Twenty
Kohno et al Release of Endothelin From Porcine Aorta
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FIGURE 1. Plotting showing standard curve of endothelin
radioimmunoassay.
Mean inhibition
of binding of
[m1)'endothelin-1 in presence of 0.5-100 pg of synthetic
endothelin-1 was calculated from five consecutive assays.
B/Bo, percent of maximum [l25I]endothelin-l bound.
pieces of these strips were used in each group. Aortic
strips were suspended in a 50-ml polypropylene tube
filled with 30 ml serum-free DMEM that contained
penicillin (100 fig/ml), streptomycin (50 pglrcA), and
aprotinin (500 kallidinogenase inactivator units/ml).
In the present study, aortic strips were suspended in
DMEM because the amount of released irendothelin from cultured porcine ECs was slightly
but significantly greater when the cell culture was
performed in serum-free DMEM than in KrebsRinger solution (unpublished observation).
Materials and Supplies
Cycloheximide, angiotensin II, and vasopressin
were purchased from Sigma Chemical Co. (St. Louis,
Missouri). DMEM was purchased from GIBCO
Labs., Life Technols., Inc. (Grand Island, New
York). Polypropylene tubes were purchased from
Becton Dickinson & Co. (Lincoln Park, New Jersey).
A gift of purified human thrombin was provided by
Sankyo Co. (Tokyo, Japan). Synthetic endothelin-1,
endothelin-2, endothelin-3, big endothelin (porcine,
22-39) antiserum, somatostatin, /3-endorphin,
human secretin, and synthetic human atrial natriuretic factor-(99-126) [human ANF-(99-126)] were
purchased from Peptide Institute Inc. (Osaka,
Japan). Endothelin-1 antiserum was purchased from
Peninsula Labs. Inc. (Belmont, California). All other
materials were obtained as described previously.13
Pharmacological Treatment
Thrombin and cycloheximide were dissolved in
sterile saline solution. Thrombin was added to 30 ml
serum-free DMEM in two concentrations of 1 and 10
units/ml, respectively. Cycloheximide was added in a
concentration of 10 /xg/ml. These agents were added
in a volume of less than 0.2% of the medium, and
both control and treated mediums were maintained
at 37° C and aerated with 95% O2 and 5% CO2. In
certain aortic strips, the endothelium was removed
5
10
100
porcine big e n d o t h e l i n (pg/tube)
1000
FIGURE 2. Plotting showing standard curve of big endothelin
radioimmunoassay. Mean inhibition of binding of [125I]big
endothelin in the presence of 5-1,000 pg synthetic big endothelin was calculated from five consecutive assays. B/Bo,
percent of maximum [mI]big endothelin bound.
mechanically by rubbing the intimal surface with a
wooden applicator stick. Aortic strips without endothelium were also suspended in serum-free DMEM
under the same conditions. At the end of the experiment, the medium was aspirated and centrifuged at
3,000g for 15 minutes, and the supernatant was
collected and stored at -80° C until the time of
radioimmunoassay.
Extraction of ir-Endothelin and ir-fljg Endothelin
Extraction of ir-endothelin and ir—big endothelin
was performed by the method previously described.12
Briefly, 10 ml of each sample was diluted with 10 ml
of 4% acetic acid. After centrifugation, the solution
was pumped at a rate of 1 ml/min, through a Sep-Pak
Cjg cartridge (Waters Chromatography Div., Millipore Corp., Milford, Massachusetts). After washing
with distilled water, the absorbed peptides were
eluted with 86% ethanol in 4% acetic acid. After
evaporation by a centrifugal evaporator (model RD31, Yamato Scientific Co., Japan), the dry residue
was dissolved in an assay buffer. The recovery rate
was calculated by adding two different quantities of
cold endothelin-1 (2 pg/ml and 20 pg/ml) and cold
porcine big endothelin (5 pg/ml and 50 pg/ml) to
serum-free DMEM. The recovery rate was 63% and
60%, respectively.
Radioimmunoassay of Endothelin
The ir-endothelin concentration was determined
by using antibody against synthetic endothelin-1 and
[125I]endothelin-l as a tracer. This antibody crossreacted 84% with endothelin-2, 5% with endothelin3, and 18% with big endothelin (porcine, 1-39),
whereas it did not cross-react with somatostatin,
/3-endorphin, human secretin, angiotensin II, vasopressin, or human ANF-(99-126).
Endothelin-1 was radioiodinated by the chloramine-T method. The radioimmunoassay was per-
720
Hypertension
Vol 15, No 6, Part 2, June 1990
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FIGURE 3. Plotting showing release of immunoreactive endothelin from porcine aortic strips. Aortic
strips with endothelium were washed twice with serum-free Dulbecco's modified Eagle's medium
(DMEM) and subsequently suspended in serum-free DMEM in the presence ( • — • ) or absence (o — o)
of 10 fig/ml cyclohadmide. Aortic strips without endothelin ( • — • ) were also suspended in serum-free
DMEM. Number of tubes is shown in parentheses; bars show SD. Hatched area is undetectable range
of immunoreactive endothelin concentration.
formed in an assay buffer of 0.01 M sodium phosphate (pH 7.4) that contained 0.05 M NaCl, 0.1%
bovine serum albumin, 0.1% Nonidet NP-40, and
0.01% NaN3. Rehydrated antiserum (100 yX) was
added to 100 /xl sample or to 100 y\ standard
endothelin-1 prepared in an assay buffer and then
incubated for 24 hours at 4° C. Approximately 15,000
cpm [12iI]endothelin-l was added to each reaction
and incubated for an additional 24 hours. After the
second 24-hour incubation, 100 yX diluted normal
rabbit serum and 100 yX diluted goat anti-rabbit
immunoglobulin G (IgG) serum were added and
again incubated for 24 hours. After the third 24-hour
incubation, the precipitate was collected by centrifugation at l,700g for 30 minutes. The supernatant was
removed by aspiration, and the pellet was counted
for 125I using a gamma counter. The effective range of
the standard curve was 1-80 pg endothelin-1 (Figure
1). The interassay variation was 13.8%, and the
intra-assay variation was 7.6%.
Radioimmunoassay of Big Endothelin
The ix—big endothelin concentration was determined using antibody against synthetic big endothelin (porcine, 22-39) and [125I]big endothelin (porcine, 1-39) as a tracer. This antibody cross-reacted
equally with big endothelin (porcine, 1-39) and big
endothelin (porcine, 22-39), 10% with big endothelin (human, 1-38) and big endothelin (human, 1838), and less than 0.1% with endothelin-1, endothe-
lin-2, and endothelin-3, whereas it did not show any
cross-reaction with somatostatin, ^-endorphin,
human secretin, angiotensin II, and human ANF(99-126).
Big endothelin (porcine, 1-39) was radioiodinated
by the chloramine-T method. The radioimmunoassay
was performed as previously described. The effective
range of the standard curve was between 10 and 600
pg per assay tube. Fifty percent intercept was 125 pg
big endothelin (Figure 2). The interassay variation
was 14.2%, and intra-assay variation was 9.6%.
Reverse-Phase High-Performance
Liquid Chromatography
Reverse-phase high-performance liquid chromatography (HPLC) was performed using an octadecylcilica column (0.46x25.0 cm, GASCHRO Co.,
Tokyo, Japan) eluted with a linear gradient of acetonitrile of 33-51% in 0.09% trifluoroacetic acid and
0.01 M NaCl with a flow rate of 1 ml/min; 0.5 ml
fractions were collected and assayed for radioimmunoassay. For chromatographic analysis of irendothelin and ir-big endothelin, 50 ml pooled
medium incubated with aortic strips was separated
and subjected to reverse-phase HPLC.
Calculations and Statistical Analysis
The statistical significance of the results was evaluated by analysis of variance, and p values were
Kohno et al
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FIGURE 4. Bar gra/?/i showing effect of thrombin (10 units/
ml, 1 unit/ml) on release of immunoreactive endothelin from
porcine aortic strips. Aortic strips were suspended in serum-free
Dulbecco's modified Eagle's medium (DMEM) alone (CON),
serum-free DMEM containing 10 units/ml thrombin (THR
[cross-hatched area], 10 units/ml), serum-free DMEM containing 1 unit/ml thrombin (THR [dotted area], 1 unit/ml), or
serum-free DMEM containing 10 units/ml thrombin and 10
liglml cycloheximide (THR+CYC). Incubation was for 6 and
12 hours. Each column represents the mean of five tubes; bars
show SD. Hatched area is undetectable range of immunoreactive endothelin concentration.
determined by Scheffe's method.14 Values were
expressed as mean±SD.
Results
Aortic strips with endothelium released irendothelin in a time-dependent manner into the
medium (Figure 3). This release was markedly inhibited when strips were incubated in the presence of 10
IMg/m\ cycloheximide. After removal of endothelium
by rubbing the intimal surface, aortic strips did not
release a detectable amount of ir-endothelin.
The addition of 10 units/ml thrombin resulted in a
significant increase in the accumulation rate of irendothelin during 6 and 12 hours (Figure 4). The
stimulatory effect of 10 units/ml thrombin was significantly greater than that of 1 unit/ml thrombin over
12 hours. Incubation of strips with 10 units/ml thrombin in combination with 10 /xg/ml cycloheximide did
not release a detectable amount of ir-endothelin into
the medium.
Release of Endothelin From Porcine Aorta
721
Aortic strips with endothelium released ir-big
endothelin in a time-dependent manner into the
medium (Figure 5). This release was markedly inhibited when strips were incubated in the presence of 10
/ig/ml cycloheximide. After removal of endothelium
by rubbing the intimal surface, aortic strips did not
release a detectable amount of ir-big endothelin.
The addition of 10 units/ml thrombin significantly
increased the accumulation rate of ir-big endothelin.
An elution profile of pooled medium extracts on
reverse-phase HPLC is shown in Figure 6. Two
components of ir-endothelin were observed, that is,
the major component eluted in the position of standard endothelin-1 and the minor component eluted
in the position of standard big endothelin (porcine,
1-39). Moreover, two components of ir-big endothelin were observed, the major component eluted in the
position of standard big endothelin (porcine, 1-39)
and another minor component eluted earlier than
porcine big endothelin (1-39) standard.
Discussion
The present study has shown that porcine aortic
strips with endothelium are able to release irendothelin into the medium in a time-dependent
manner. This release was markedly inhibited by protein synthesis inhibitor cycloheximide. Several recent
reports indicate that cultured ECs release irendothelin in a time-dependent manner.10'15'16 These
studies using cultured ECs, however, do not necessarily mean that endothelin is released from aortic endothelium in vivo. The present experiment using aortic
strips can support the hypothesis that endothelin is
released slowly but continuously into the extracellular
space in vivo. This hypothesis seems to be supported
by recent reports17-19 that ir-endothelin is present in
human plasma. Our experiment also shows that, in
addition to this basal release, there is an extra release
that can be stimulated by thrombin. This finding seems
to be compatible with the recent report20 that thrombin enhances the release of ir-endothelin from cultured porcine aortic ECs.
In the present study, we have also shown that
porcine aortic strips with endothelium release ir-big
endothelin in a time-dependent manner. This release
was markedly inhibited by cycloheximide and was
significantly stimulated by thrombin. Reverse-phase
HPLC coupled with radioimmunoassay shows that
both endothelin-1 and big endothelin are released
from porcine aortic endothelium. C-terminal fragment
(22-39) of big endothelin as well as endothelin and
big endothelin, however, can be released from aortic
strips because the antibody used in this study crossreacts equally with big endothelin (1-39) and big
endothelin (22-39). Actually, recent studies21-22 demonstrated that C-terminal fragment (22-39) of big
endothelin releases into the culture medium of ECs.
Although the exact mechanism of endothelin and
big endothelin release by endothelium remains to be
clarified, our data show that aortic strips with endothelium release endothelin and big endothelin into
Vol 15, No 6, Part 2, June 1990
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FIGURE 5. Plotting showing
release of immunoreactive big
endothelin from porcine aortic
strips and effect of thrombin.
Aortic strips with endothelium
were washed twice with serumfree Dulbecco's modified Eagle's
medium (DMEM) and subsequently suspended in serum-free
DMEM in the presence ( • — • )
or absence (o —o) of 10 fig/ml
cycloheximide. Aortic strips were
also suspended in serum-free
DMEM containing 10 units/ml
thrombin (o — 0 ). Aortic strips
without endothelium (A —A)
were suspended in serum-free
DMEM. Number of tubes is
shown in parentheses; bars show
SD. Hatched area is undetectable range of immunoreactive
big endothelin
concentration.
tp<0.05 versus aortic strips suspended with serum-free DMEM
alone.
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FIGURE 6. Plotting showing reverse-phase high-performance liquid chromatography profile of immunoreactive
endothelin and big endothelin in medium extracts. Medium samples were pretreated with Sep-Pak C,s as described
in text. Elution positions of standard endothelin-1 and big endothelin (porcine, 1-39) are shown by arrows.
Kohno et al
the extracellular space and that these releases can be
stimulated by thrombin. Further studies will be necessary to clarify the role of big endothelin and to
determine whether this low concentration of endothelin and big endothelin could have a significant
influence on vessel tone in vivo.
References
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Release of Endothelin From Porcine Aorta
723
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KEYWORDS • endothelin • thrombin • aorta • endothelium
Release of immunoreactive endothelin from porcine aortic strips.
M Kohno, K Yasunari, K Murakawa, K Yokokawa, T Horio, T Fukui and T Takeda
Hypertension. 1990;15:718-723
doi: 10.1161/01.HYP.15.6.718
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