378
Effect of Carbon Dioxide and Oxygen on
Endothelin Production by Cultured Porcine
Cerebral Endothelial Cells
Satonobu Yoshimoto, MD, DMSc; Yasuki Ishizaki, MD, PhD;
Tomio Sasaki, MD, DMSc; and Sei-itsu Murota, PhD
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We have previously reported the production of endothelin, a potent vasoconstrictor peptide, by
porcine cerebral microvessel endothelia and suggested its important role in the regulation of
local blood flow within the brain. In our present study, radioimmunoassay with anti-porcine
endothelin antiserura revealed that endothelin, produced by cerebral microvessel endothelia
grown on a filter, is released mainly to the basement membrane side, not the vascular lumen
side. This finding suggests that endothelin constricts arterioles locally where it is produced by
endothelia. We also found that cerebral microvessel endothelia produce less endothelin under
low oxygen pressure and more endothelin under low carbon dioxide pressure. Our results
suggest that endothelin has a role in the regulation of cerebral bloodflowin response to oxygen
and carbon dioxide pressure. (Stroke 1991;22:378-383)
V
ascular endothelia have been suggested to
play important roles in the regulation of
blood flow in various tissues.1 In 1982 and
1983, De Mey and Vanhoutte2-3 reported vasoconstriction dependent on or enhanced by the endothelium. Since then, vasoconstriction dependent on endothelial cells with various chemical stimuli has also
been reported. 4 - 6 In 1987, O'Brien et al7 suggested
the presence of a vasoconstrictor peptide in the
conditioned medium from cultured bovine aortic and
pulmonary arterial endothelial cells and called it an
endothelium-derived constricting factor. In 1988,
Yanagisawa et al8 isolated a vasoconstrictor peptide
(endothelin) from the supernatant of cultured endothelia of porcine aorta, and endothelin is now believed to be the most likely candidate for endothelium-derived constricting factor. Endothelin was
found to constrict intracranial arteries in vitro and in
vivo9 and is reported to have a potent microvascular
constrictive effect.10 Since the work of Kety and
Schmidt,11 it is believed that the brain demands a
critical and constant blood supply to maintain neural
function. Neural, chemical, and metabolic factors
From the Department of Neurosurgery (S.Y., T.S.), Faculty of
Medicine, University of Tokyo and the Section of Physiological
Chemistry (Y.I., S-i.M.), Faculty of Dentistry, Tokyo Medical and
Dental University, Tokyo, Japan.
Address for correspondence: Satonobu Yoshimoto, MD, Department of Neurosurgery, Faculty of Medicine, University of
Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan.
Received April 2, 1990; accepted November 7, 1990.
influence cerebral blood flow, as do changes in
arterial blood pressure.12
We recently reported endothelin production by
brain microvessel endothelia and suggested an important role for this substance in the regulation of
local cerebral blood flow.13 In our present study, we
examine the polarity (luminal versus abluminal) of
endothelin release and the effect of oxygen and
carbon dioxide pressure on endothelin production by
cultured cerebral microvessel endothelial cells to
corroborate the possibility that endothelial cells are
involved in the regulation of cerebral blood flow
through the production of endothelin.
Materials and Methods
Primary cultures of brain microvessel endothelial
cells were prepared after the method of Bowman et
aji4,i5 w jth slight modifications as reported previously.13 The brains of two adult pigs were transported
from a slaughterhouse to the laboratory s i hour
after sacrifice in ice-cold tissue culture medium containing antibiotics. After removing the choroid plexuses and diencephalon, we cleaned the cerebral
cortical tissues of meninges and superficial blood
vessels and minced the tissue into 1-2-mm cubes.
The tissue was incubated in 400 ml minimal essential
medium (MEM) (GIBCO, Grand Island, N.Y.) containing 0.5% dispase at 37°C for 3 hours. After
centrifugation at l.OOOg for 10 minutes, the pellet was
collected and resuspended in 200 ml MEM and
centrifuged again under the same conditions. The
obtained pellet was resuspended in 200 ml MEM
Yoshimoto et al Cerebral Circulation and Endothelin
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containing 13% dextran (Dextran T 70, Pharmacia,
Uppsala, Sweden) and centrifuged at 5,800g for 10
minutes to remove the cellular debris and myelin
components. The pellet was resuspended in 60 ml
MEM and filtered through 300-/im nylon mesh.
From the filtrate, microvessels were collected by
centrifugation at l.OOOg for 10 minutes. The microvessels were then incubated in 4 ml medium
containing 1 mg/ml collagenase/dispase (Boehringer
Mannheim, Mannheim, FRG) for 10 hours to remove the basement membrane and most pericytes.
Microvessels were centrifuged at l,000g for 10
minutes, and the pellet was resuspended in 2 ml
medium. The suspension was then layered on 8 ml
Percoll (Pharmacia) gradient that had been prepared
previously by centrifugation at 26,000g for 1 hour; the
layer of berry-like groups of endothelia were obtained by centrifugation at 600g for 10 minutes. The
cells were resuspended in 10 ml medium and collected by centrifugation at l,000g for 10 minutes. The
cells were seeded on 2.0 cm2x24-well culture dishes
or on collagen-coated filters (Intercell, 0.45 //.m,
Kurabo, Osaka, Japan) placed in 24-well culture
dishes and were incubated at 37°C in MEM containing 10% fetal bovine serum (FBS) (Whittaker Bioproducts, Walkersville, Md.), 100 /ig/ml penicillin,
100 ^.g/ml streptomycin, 2.5 /ig/ml amphotericin B,
and 20 units/ml heparin in a 95%/5% mixture of
atmospheric air and CO2. After 4-6 days, a confluent
cell monolayer was obtained. The cells were identified by their incorporation of acetylated low density
lipoprotein labeled with l,l'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate (Paesel,
Frankfurt, FRG). 16 The conditioned medium of the
primary cultured cells after confluence was used for
radioimmunoassay.
To determine the polarity of endothelin release,
we used endothelia cultured on the collagen-coated
filters.17 After confluence, the cells were placed in
new dishes. To equalize the hydrostatic pressure of
both sides when 150 p\ medium was put into each
side, 600 /i\ of the lower part was replaced by
Bio-Resin (Pile B) (Shofu, Kyoto, Japan), which is
nontoxic and is made for dental splints or artificial
surgical bone. The cells were then placed in the
incubator for 2 hours. This incubation time was
chosen in an attempt to allow production of enough
endothelin and to minimize the nonspecific movement of endothelin through the cell-filter complex.
The medium on each side of the cell-filter complex
was collected and subjected to radioimmunoassay for
endothelin. Tightness of the cell-filter complex was
certified by examining spectrophotometrically the
permeability of a 0.1 g/ml bovine serum albumin
(Wako, Osaka, Japan)-0.02% Evans blue complex1*
placed on the upper side. The amount of Evans blue
filtered was about Vu of that applied to the upper
side. (The mean±SEM value of the upper side was
781 ±56.78 [n=4] and that of the lower side was
60.5±2.54 after 2 hours' incubation.)
379
For the low-oxygen experiments, confluent endothelia from the culture dishes were used. After
medium exchange with 200 jil culture medium, the
cells were cultured under 25-30 mm Hg (approximately 4%) O2 or 80 mm Hg (approximately 10%) O2
for 0-6 hours; 20% (approximately 155 mm Hg) O2
was used as the control. The actual oxygen content of
the conditioned medium was measured using a blood
gas analyzer (Acid-Base Laboratory 4, Radiometer,
Copenhagen, Denmark) immediately after sampling.
For the 4% O2 experiment, two chamber boxes
(Modular Incubator Chamber, St. Hart Container
Corp., Cleveland, Ohio) were used. Each chamber
has an airtight cavity, which can be opened, and two
clampable rubes. Through the inlet tube, the chamber was flushed with a 2.5 1/min 5%/95% CO2/N2 gas
mixture for 15 minutes. After both tubes were
clamped, the chambers were placed in the incubator
at 37°C. Before the experiment, the oxygen content
was checked and the PO2 was verified to be 25-30
mm Hg at 3, 6, and 12 hours' incubation after clamping at the same condition. After medium exchange,
the culture dishes were placed in the chamber. After
closing the chamber, it was flushed as above and
placed in the incubator. Media were collected 3, 6,
and 12 hours after medium exchange and were
subjected to radioimmunoassay for endothelin. In a
separate experiment, 1 and 2 hours' incubations were
performed with 20% O2 as the control. At the end of
each experiment, cell viability was checked by the dye
exclusion method using erythrosin B, and almost no
cells were dyed at 4% O2.
Endothelin production by the microvessels themselves was also measured. Microvessels taken from
half of each porcine brain before treatment with
collagenase/dispase were incubated in 5 ml MEM
containing 10% FBS under 4% or 20% O2. After 19
hours' incubation, the media were collected and
subjected to radioimmunoassay for endothelin.
For the 10% O2 experiment, the incubator, which
can control oxygen content, was used. After medium
exchange the culture dishes were placed in the
incubator, and conditioned media were obtained 1, 2,
3, and 6 hours later and subjected to radioimmunoassay for endothelin. All procedures were undertaken in the incubator.
For the low-carbon dioxide experiments, two incubators, which can control both carbon dioxide and
oxygen contents, were used. Media were placed in
each incubator for 24 hours before the experiment
(incubator 1: 5% CO2 and 19% O2 [normal control];
incubator 2: 2.5% CO2 and 19% O2). After medium
exchange with 200 /xl culture medium, confluent
endothelia from the culture dishes were placed in
each incubator. Conditioned media were obtained
after 15, 30, 60, and 120 minutes' incubation.
Amersham's Endothelin-1,2 [125I]Assay System
(Amersham, U.K.), by which the concentration of
endothelin-1,2 can be measured in the range 0.5-64
fmol/tube, was used as the manufacturer recommends. Briefly, 100 jtl of each conditioned medium
380
Stroke
Vol 22, No 3 March 1991
FIGURE 1.
A: Phase-contrast
micrograph of cultured endothelium of porcine brain microvessel B: Almost all cells were identified as endothelial cells by
incorporation of acetylated low
density lipoprotein.
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was pipetted into tubes, 100 /AI endothelin-1,2 antiserum was added to every tube, and the tubes were
incubated for 24 hours at 4°C. Then, 100 /xl [125I]endothelin-1,2 was added to all tubes, and they were
incubated for 24 hours at 4°C. Then, 250 /AI of a
second antibody was added to each tube, and they
were incubated for 10 minutes at room temperature.
The pellet, which contained the second antibodybound fraction, was obtained after centrifuging at
l,500g for 10 minutes and subjected to gamma scintillation counting.
Results are given as mean±SEM. We compared
endothelin production under different oxygen and
carbon dioxide pressures using the t test.
Results
The cells used are shown in Figure 1A. Almost all
cells incorporated acetylated low density lipoprotein and thus were identified as endothelial cells
(Figure IB).
When the cells were grown on a filter (Figure 2),
endothelin was released both apically and basally,
but endothelin release from the basal side was about
twice than from the apical side (apical endothelin
release: 25.3±1.0 fmol/ml; basal endothelin release:
48.9±6.9 fmol/ml; n=4). The background level of
endothelin in culture medium containing 10% FBS
was 51.8±1.9 fmol/ml (n=3).
Endothelin production under 4% O2 and 20% O2
was proportionate to the incubation time, and production under 4% O2 was always significantly lower
(p<;0.01) than that under 20% O2 (Figure 3). Endothelin production under 4% O2 was significantly
lower after as little as 1 hour's incubation (Figure 3,
150//! (Lower)
150/// (Upper)
25.3±1.0fmo|/roi
600///
of Bio Resin
48.9±6.9fmol/m*
endothelin
(mean±SE)(n=4)
FIGURE 2. Schema of cultured
cell-filter complex. Same volume of medium (150 \d) was
placed into both sides; 600 ul
Bio-Resin was placed to equalize
hydrostatic pressure of both
sides. Mean±SEM
endothelin
concentration in apical side was
25.3+1.0 fmol/ml and that in
basal side was 48.9±6.9 fmoll
ml Similar result was obtained
in another experiment using different cell line.
Yoshimoto et al Cerebral Circulation and Endothelin
381
2 hours
FIGURE
3.
Graph
of
the
mean ±SEM endothelin production/200 \d medium/1.5x10'
endothelia under 4% O2 (x)
and 20% O2 (•). Similar results
were obtained in three other experiments using different cell
lines.
15001200900-
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6003000
12 hours
inset). Contrary to this, endothelin production under
10% O2 was similar to that under 20% O2 from 1
through 6 hours (data not shown).
The amount of endothelin in medium conditioned
by the microvessels themselves after 19 hours' incubation was 83.8 fmol/ml under 4% O2 (n=2) and
186.3 fmol/ml under 20% O2 (n=2). The background
level of endothelin in culture medium containing
10% FBS was 54.9±1.8 fmol/ml (n=3). Judging from
this result, microvessels themselves produced less
endothelin under 4% O2 than under 20% O2.
Endothelin production under 2.5% and 5% CO2
was examined from 15 through 120 minutes. Production was almost proportionate to the incubation time
(Figure 4). However, endothelin production under
2.5% CO2 was significantly higher than that under
5% CO2 at 60 and 120 minutes (p^O.Ol).
All these results differed slightly among cell lines, but
the same tendencies were observed in all cell lines.
Discussion
Recently, the presence of three isopeptides of
endothelin, the "classical" endothelin (ET-1),
[Trp«,Leu7]endothelin (ET-2), and [Thr2, Phe4,Tyr*,
Lys7,Tyr14] endothelin (ET-3), has been suggested by
analysis of the human genome.19 To date, however,
only the presence of ET-1 has been confirmed by its
isolation from the conditioned medium of cultured
aortic endothelial cells.8 It is also reported that
vascular endothelial cells express only ET-1. 19
Therefore, the endothelin detected in our study is
presumed to be ET-1.
We demonstrated that endothelia secrete endothelin more into the basal side, presumed to be the
ftnol/o/
2.596CO,
300
5% CO,
200-
100-
15
30
60
120min
FIGURE
4.
Graph
of
the
mean±SEM endothelin production/200 id medium/1.5xlOs
endothelia under 2.5% CO2 f x)
and 5% CO2 (•). Similar result
was obtained in another experiment using different cell line.
382
Stroke Vol 22, No 3 March 1991
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abluminal side, than into the apical side, presumed to
be the luminal side. This suggests that endothelin,
which is produced locally by cerebral microvessel
endothelia, is released mainly not into the vascular
lumen, but to the basement membrane side. This
suggests that endothelin constricts arterioles locally
where it is produced by endothelia.
There have been many reports that hypoxia dilates
brain pial arterioles, and this effect is pronounced at
low oxygen pressures in the range 20-30 mm Hg.20-22
Our results are compatible with this phenomenon.
Our results are also compatible with the report that
hypoxia dilates cerebral blood vessels via a local
mechanism.20 When PO2 in the brain is low, endothelia produce less endothelin, resulting in less constriction of the arterioles and augmenting blood flow
in the brain. This may be due simply to a low
metabolic rate under low oxygen pressures. Even if
this is the case, it results in less endothelin production and thus an increase in the blood flow. From our
results, endothelin production under 10% and 20%
O2 did not differ. This might be explained by the fact
that endothelia in situ are usually under about 80
mm Hg (approximately 10%) O2; therefore 10% O2 is
not hypoxic to endothelia.
Endothelia produced more endothelin under 2.5%
CO2 than under 5% CO2. This result is compatible
with reports that hypocapnia induces cerebral arteriole constriction.22-24 Hypocapnia is usually accompanied by alkalosis. In our case, pH of the medium,
which was incubated under 2.5% CO2, was 7.62 (pH
of the 5% CO2 medium was 7.32). Although it
remains undetermined whether the increase in endothelin release by cerebral microvessel endothelia is
due to alkalosis or hypocapnia, our results may
explain how cerebral arterioles constrict under hypocapnia or alkalosis.
The effect of endothelin is prolonged8 while the
actions of hypocapnia and hypoxia are much quicker
and readily reversible in vivo.20'23 Also, our data were
obtained from cultured cells; thus, they may not be
directly related to the physiologic roles of endothelin.
However, as the actual function, concentration, or
degradation of endothelin in situ are still undetermined, our observations may reveal some aspects of
the roles endothelin plays in vivo.
It is said that the brain demands a critical and
constant blood supply to maintain neural function.
PO2 and PCO2 are two of the most important factors
that control cerebral blood flow. Along this line, we
suggest for the first time that endothelin has a role in
the regulation of cerebral blood flow in response to
oxygen and carbon dioxide pressure.
Acknowledgments
We thank Ms. Reiko Matsuura and Ms. Arisa Mori
for their excellent technical assistance, and we thank
members of Tokyo Metropolitan Slaughterhouse for
giving us great facilities to get fresh samples.
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KEY WORDS
cerebral blood flow
endothelin
Pigs
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Effect of carbon dioxide and oxygen on endothelin production by cultured porcine cerebral
endothelial cells.
S Yoshimoto, Y Ishizaki, T Sasaki and S Murota
Stroke. 1991;22:378-383
doi: 10.1161/01.STR.22.3.378
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