Proc. Nadl. Acad. Sci. USA
Vol. 85, pp. 9797-9800, December 1988
Pressor effects of circulating endothelin are limited by its removal
in the pulmonary circulation and by the release of prostacyclin
and endothelium-derived relaxing factor
(lung/guinea pig/rat/icosanoids/cyclooxygenase inhibitors)
GILBERTO DE Nucci, ROGER THOMAS, PEDRO D'ORLEANS-JUSTE, EDSON ANTUNES, CLAIRE WALDER,
TIMOTHY D. WARNER, AND JOHN R. VANE
The William Harvey Research Institute, St Bartholomew's Hospital Medical College, Charterhouse Square, London EC1M 6BQ, United Kingdom
Contributed by John R. Vane, September 26, 1988
boxane A2 from guinea pig or rat isolated lungs and endothe-
lium-derived relaxing factor in the perfused mesentery of the
rat. Endothelin is also substantially removed by the pulmonary
circulation ofthe rat in vitro and in vivo and by guinea pig lungs
in vitro. In the rat, the effects ofendothelin on the blood pressure
vary from pressor (in pithed rats) to purely depressor in
anesthetized rats where the resting blood pressure is high. It
therefore4iasthe characteristics of a local pressor hormone,
rather than a circulating one.
Endothelin releases prostacyclin and throm-
The endothelial cell (EC) is known to release vasoactive
substances such as prostacyclin (PGI2) (1) and endothelium-
derived relaxing factor (EDRF) (2), recently identified as
nitric oxide (3). Release of endothelium-dependent vasocon-
strictor factors has been observed in response to various
chemical and physical stimuli such as norepinephrine (4),
thrombin (4), hypoxia (5, 6), increased transmural pressure
(7), and mechanical stretch (8).
Masaki and his colleagues (9) have recently characterized
from cultures of porcine aortic ECs a 21-amino acid peptide,
which they called endothelin (ET). In the chemically dener-
vated rat, porcine ET is the most potent pressor substance
yet described, with a long duration ofaction. They suggested
that ET directly activates dihydropyridine-sensitive calcium
We report here that apart from its vasoconstrictor activity,
ET can release potent vasodilator substances such as PGI2
and EDRF and is also removed by the pulmonary circulation.
MATERIALS AND METHODS
Superfusion Bioassay. Spiral strips of de-endothelialized
vascular smooth muscle from the rabbit (mesenteric artery,
celiac artery, carotid artery, aorta, jugular vein, mesenteric
vein) and other smooth muscle preparations (guinea pig
trachea, guinea pig ileum, rat stomach strip, rabbit duode-
num) were mounted in a cascade (10) and superfused at 5
ml-min-1 with Krebs-Ringer solution containing indometh-
acin (5.6 ,M). Agonists such as ET (1-50 pmol), bradykinin
(1-10 pmol), substance P (1-10 pmol), and angiotensin 11(1-
10 pmol) were injected over the assay tissues.
Isolated Lungs. Male Dunkin-Hartley guinea pigs (300-400
g) or male Wistar rats (200-300 g) were anesthetized with
sodium pentobarbital (Sagatal, 70 ,umol kg-', i.v.) and a
thoracotomy was performed. The pulmonary artery and the
trachea were cannulated and the lungs were removed and
placed in a warm chamber. The lungs were perfused at S
ml-min-' via the pulmonary artery with oxygenated (95% 02/
5% CO2) and warmed (370C) Krebs-Ringer solution (11). The
lungs were left to stabilize for 30 min and ET was infused for
3 min at a flow rate of 0.1 ml-min-' to achieve a final
concentration of 1 or 10 nM. The effluent from lungs was
collected and analyzed by RIA for 6-oxoprostaglandin F1l
(6-oxo-PGF1,) and thromboxane (TX) B2 as measures of
prostacyclin and TXA2 release (12). The removal of ET was
calculated by comparing the contractions ofthe assay tissues
in response to infusions of ET directly over the tissues with
those in response to infusions given through the lungs (13).
Isolated Mesentery. The rat isolated perfused mesentery
was prepared from male Wistar rats (200-300 g) pretreated
with heparin (1000 units-kg-', i.p.) (14). The mesenteric bed
was perfused at 5 ml-min-1 with warmed (370C) and gassed
(95% 02/5% C02) Krebs-Ringer solution, which contained
indomethacin (5.6AM)and sometimes albumin (0.5% wt/
vol). Vascular tone in the bed was increased by an infusion
of norepinephrine, U46619, 9,11-dideoxy-9a,11a-methano-
epoxyprostaglandin F2a (0.03-1.5 ,uM), or methoxamine (30-
100AM)to produce an increase in perfusion pressure from
15-22 to 35-130 mmHg. In experiments in which the ECs
were removed from the mesentery by infusing sodium de-
oxycholate (2.4 mM), the Krebs-Ringer solution contained
albumin (0.5%) to minimize subsequent edema (15).
ECs. ECs were isolated by treatment ofbovine aortae with
0.02% (wt/vol) collagenase. Cells were grown to confluence
in plastic vessels and then removed by treatment with 0.05%
(wt/vol) trypsin and seeded onto Cytodex 3 microcarrier
beads. The beads were stirred for 3-7 days until the cells
became confluent and were then packed into a jacketed
column and perfused (5 ml-min-1 at 370C) with gassed (95%
02/5% C02) Krebs-Ringer solution, which containedsuper-
oxide dismutase. EDRF was bioassayed on a cascade of
rabbit aortae (16-18).
Blood Pressure. Male Wistar rats (250-300 g) were anes-
thetized with thiobutabarbital (Inactin) (0.5 mmol'kg-1) or
pithed under halothane anesthesia. The right atrium and the
left ventricle were cannulated via the right common carotid
artery and left jugular vein, respectively. Peripheral blood
pressure and heart rate were measured on a Grass model 7D
polygraph from a femoral artery cannula. In the anesthetized
animals, ET (0.01-1 nmol kg-1) was injected in volumes of
either 1.0 or 0.3 ml-kg-' alternately into the arterial or venous
sides of the circulation. In pithed rats, cumulative dose-
response curves to both intraarterial (i.a.) and i.v. ET were
made over a dose range of 0.2-2 nmol kg-1.
Bradykinin, substance P, angiotensin II, acetylcholine
(ACh), norepinephrine, methoxamine, sodium deoxycholate,
indomethacin, albumin, piroxicam, methylene blue, and
Abbreviations: PGI2, prostacyclin; EDRF, endothelium-derived re-
laxing factor; EC, endothelial cell; ET, endothelin; TX, thrombox-
ane; 6-oxo-PGFia, 6-oxoprostaglandin Fia; ACh, acetylcholine.
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Medical Sciences: de Nucci et al.
angiotensin II (All), substance P (SP), and bradykinin (BK) in
various smooth muscle preparations. RbJV, rabbit jugular vein;
RbMesV, rabbit mesenteric vein; RbMesA, rabbit mesenteric artery;
RbCoA, rabbit celiac artery; RbCrA, rabbit carotid artery; RbA,
rabbit aorta; RbD, rabbit duodenum; GPT, guinea pig trachea; RSS,
rat stomach strip; RC, rat colon.
A representation of the pharmacological profile of ET,
hemoglobin were obtained from Sigma. Inactin (thiobutabar-
bital) was obtained from Byk Guiden Pharmazeutika. Sa-
gatal (sodium pentobarbital) was obtained from May & Baker
(Dagenham, U.K.) Captopril was a gift from B. H. Boeree
(Squibb Europe); [3H]6-oxo-PGFia and [3H]TXB2 were pur-
chased from New England Nuclear. Synthetic ET (porcine;
Peptide Research Institute, Osaka, Japan) was agenerous gift
of T. Masaki (Tsukuba University, Japan). Bay K 8644, the
methylphenyl)-pyridine-5-carboxylic acid methyl ester was a
gift ofF. Seuter (Bayer, F.R.G.). The 6-oxo-PGFia and TXB2
were kindly provided by J. Pike (Upjohn). The icosanoid
antisera were kindly provided by J. A. Salmon (Wellcome).
ET (1 or 10 nM) added to rabbit or human platelet-rich plasma
did not induce aggregation, nor did it affect the aggregation
induced by ADP, collagen, or arachidonic acid (n = 3). The
activity of ET on rabbit jugular vein was not affected by
incubation in rabbit or human platelet-poor plasma (n = 3) or
in rabbit whole blood (n=3) forup to 90 min at 370C, showing
that it was resistant to degradation by plasma peptidases and
Isolated Smooth Muscle. Venous strips were more sensitive
to ET than arterial strips (Fig. 1) and, in particular, the rabbit
jugular vein and mesenteric vein were contracted by as little
as 0.5-2.5 pmol of ET in a reproducible and dose-dependent
way. The rat stomach strip was also a useful bioassay tissue
in that contractions produced by ET (5-15 pmol) returned to
baseline faster than those of venous strips and much faster
than those of arterial ones.
Release of Icosanoids from Lungs. ET (1 or 10 nM) infused
for 3 min through isolated lungs of guinea pigs induced a
sustained release of PGI2 and TXA2 (Fig. 2). In rat lungs, ET
(10 nM) induced a much stronger release of PGI2 than TXA2
(6.6± 2.1 ng ml-l and 0.4 ± 0.2 ng ml-1 for 6-oxo-PGFia and
TXB2, respectively; n=4).
Removal of ET by Lungs. ET (1 nM) infused through the
pulmonary circulation of guinea pig isolated lungs (treated
with indomethacin to prevent icosanoid release interfering
with the bioassay) was substantially removed, so that only
40%±3.6% (n=4) ofthe amount infused was detected in the
effluent (Fig. 3). The removal of ET was not affected by an
angiotensin-converting enzyme inhibitor (captopril) given in
sufficient concentrations (10 ,uM) to inhibit substantially the
inactivation of bradykinin (19).
Release of EDRF. ACh provoked dose-dependent vasodi-
latations ofthe rat isolated perfused mesentery due to EDRF
release, for after removal of the ECs with sodium deoxycho-
late or in the presence of oxyhemoglobin (30 ,uM), vasodi-
latation induced by ACh was suppressed (n > 30; Fig. 4). ET
(1-10 pmol) also induced dose-dependent vasodilatations
through EDRF release (n=18). In a further 12 experiments,
ET (1 nM)
ofboth PG12 and TXA2 [measured by radioimmunoassay as6-oxo-PGF1, (m) and TXB2 (e)]. The release ofboth icosanoids continued afterthe
infusion was terminated (n=3). The SEM values (± 5-15%) for the 6-oxo-PGFia concentrations were omitted for the sake ofclarity. Therelease
of icosanoids was confirmed by bioassay.
ET releases PGI2 and TXA2 from isolated lungs of guinea pigs. ET (1 or 10 nM) infused for 3 min induced dose-dependent release
Proc. Natl. Acad. Sci. USA 85(1988)
Proc. Natl. Acad. Sci. USA 85 (1988)
from guinea pig lungs treated with indomethacin (5.6gM)superfused
a rat stomach strip (RSS). ET (1 nM) infused through the lungs (TL)
induced a contraction ofthe RSS smallerthan that induced by ET (0.5
nM) infused over the tissues (OT), indicating a removal of >50%o in
a single passage. Bradykinin (3 nM) was removed by the lungs as
described (5), whereas iloprost (ILO) was not. Similar results were
obtained in four other experiments.
The pulmonary circulation removes ET. The effluent
EDRF release was not observed, probably because the
constrictor effects ofET were dominant. At higher doses, ET
provoked sustained increases in perfusion pressure (n = 30;
Fig. 4). After removal of the ECs (n = 6) or in the presence
of oxyhemoglobin (30AM;n = 4), ET (1-10 pmol) induced
only sustained vasoconstriction. In preparations in which no
methoxamine was infused to raise the perfusion pressure, ET
(1-100 pmol) induced dose-dependent vasoconstrictions,
which were substantially potentiated by the removal of the
endothelial cells (n = 8) or by the presence ofoxyhemoglobin
(30,M; n = 5; Fig. 4) or methylene blue (100AM;n = 5). Bay
K 8644 (1-300 pmol) did not release EDRF in the rat isolated
mesentery (n = 3). ET (1-50 pmol) or Bay K 8644 (0.27-27
nmol) did not release EDRF or PGI2 when injected through
columns of bovine aortic ECs (n = 4).
400 4000 6 60 1
tery. (A) To assess the contribution of EDRF to the vascular
responses, oxyhemoglobin (30 ,uM) was infused through the mesen-
tery. ACh-induced dose-dependent vasodilatations were mediated via
release of EDRF, for they were abolished by oxyhemoglobin. ET at
lower doses also induced vasodilatations, which were abolished by
oxyhemoglobin. Responses to sodium nitroprusside (NaNP) were
only slightly attenuated. (B) In preparations in which no tonus was
induced, ET induced vasoconstrictions that were strongly potentiated
by infusion of oxyhemoglobin.
ET releases EDRF in the rat isolated perfused mesen-
pithed rat preparations. (A) Larger transient depressor responses are
shown after i.a. injection rather than after i.v. injection of ET (0.3
nmol kg-1) in an anesthetized rat with a high resting arterial blood
pressure (185/130 mmHg) following i.a. or i.v. injection. The order
of injections did not affect the results. (B) ET (0.3 nmol kg-1, i.v. or
i.a.) producing transient depressor followed by more sustained
pressor effects in an anesthetized rat preparation with a lower resting
arterial blood pressure (130/85 mmHg). (C) Upper trace shows the
cumulative dose-response curve to ET given i.a., whereas the lower
trace describes a comparable dose-response curve to ET given i.v.
Note that in all instances i.v. injections produced smaller effects than
those given into the arterial circulation.
Blood pressure responses to ET in anesthetized and
Blood Pressure Measurements. Surprisingly, in anesthe-
tized rats ET produced dose-dependent depressor responses
(Fig. SA). These reductions in peripheral blood pressure were
most likely due to vasodilatation, for there were no apparent
effects on the heart. However, as shown in Fig. 5, these
depressor responses were smaller when ET was given i.v.
than when the peptide was injected into the left ventricle.
When the basal blood pressure was lower, the depressor
responses were followed by dose-dependent and sustained
increases in blood pressure (30 min following 1 nmol kg-1,
i.a.; Fig. 5B).
In pithed rats with low resting blood pressure of 48 ± 1.3
mmHg (n = 5), ET produced dose-dependent and prolonged
pressorresponses (Fig. 5C) and regularly increaseddp/dtmax,
suggesting venoconstriction. Again, i.v. administration ofET
caused a smaller pressor response than when itwas given into
the arterial circulation.
In the pithed rat, a second injection of ET (1 nmol kg-1)
gave a similar effect to the first (41.7 ± 9.8 and 45.3 ± 13.2
mmHg, respectively; n = 5). However, indomethacin (14
Amol-kg-1, i.v.) given before the second injection brought
about an enhancement of the pressor response to 78.3 ± 9.2
mmHg (n = 6; Fig. 6). A similar potentiation was seen with
the cyclooxygenase inhibitor piroxicam (15
control pressor response to ET (1 nmol kg-1) was 31 ± 9.6
mmHg, whereas after piroxicam it was increased to 72.7 +
9.9 mmHg (n = 4).
mol kg-l). The
ET is the most potent pressor agent yet described (9). Our
results indicate that ET can also release potent vasodilator
substances such as PGI2 [as shown in guinea pig (20) and rat
lungs] and EDRF [as shown in the rat isolated perfused
Medical Sciences: de Nucci et al.
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Medical Sciences: de Nucci et al.
the pithed rat. Overall, in six experiments, ET (1 nmoilkg-1) injected
i.v. induced an increase in blood pressure of 43.3
6). Indomethacin (Indo; 14jumol-kg-1)was administered after the
blood pressure returned to control values. A second dose of ET (1
nmol kg-1) induced an increase in bloodpressureof78.3
approximately twice that induced by the firstchallenge (n = 6). Similar
results were obtained withpiroxicam (15 umolkg-1;n = 4).
ET and Bay K 8644 (22) did not release EDRF orPGI2from
a colump of bovine aortic ECs grown on microcarrier beads
in culture. Since such cells are known to lose some receptors
(such as those forACh or5-hydroxytryptamine), it is possible
that they also lack functional voltage-dependent calcium
channels, or else these channels are not relevant for the
release ofEDRF. Indeed, the finding that Bay K 8644 did not
induce release of EDRF in the rat isolated perfused mesen-
tery, whereas ET does, would reinforce this latter interpre-
ET is stable in plasma and whole bloQd, which adds
physiological relevance to the disappearance ofET observed
in the lung circulation in vitro and in vivo. Such extensive
pulmonary removal (>50% in a single passage) indicates that
ET would be cleared from the circulation in three to five
circulation times, or 1-2 min. Thus, the long-lasting pressor
responses must be due to ET binding to, and continuously
activating, vascular smooth muscle. From our results, it is
not possible to say whether the'disappearance is due to
metabolic inactivation or to an uptake system. However, the
finding that captopril had no action on the disappearance of
ET indicates that ET is not a substrate for angiotensin-
ET induced release of PGI2 and TXA2 in guinea pig lungs
and mainly PG12 in rat lungs. Such a difference in profile of
icosanoid release between guinea pigs and rats has been seen
with other substances (23). The results obtained with the
cyclooxygenase inhibitors indomethacin and piroxicam in
rats in vivo are consistent with the release ofcyclooxygenase
products observed in this model. Indomethacin strongly
augmented the pressor activity of ET, indicating that a
prostaglandin, (probably PGO2) was indeed limiting the pres-
sor activity ofET. Interestingly, the vasodilator effects ofET
in anesthetized rats with high blood pressure were not
attenuated by indomethacin, suggesting that these were due
mainly to release of EDRF.
Thus, ET has the characteristics ofa local hormone, which,
when released by the EC, will constrict the underlying
Indomethacin potentiates the pressor response to ET in
6.1 mmHg (n =
vascular smooth muscle. Circulating ET can also release
vasodilator substances such as PG12 and EDRF. One inter-
pretation of these results is that ET released abluminally by
ECs acts locally on the underlying smooth muscle causing
vasoconstriction, whereas the pressor activity of any ET
reaching the circulating blood is limited by the release ofPGI2
and EDRF and by inactivation in the lungs. It is noteworthy
that ET was more potent as a constrictor on venous than
arterial smooth muscle. In addition, the increase in dp/dtmax
suggests venoconstriction in these animals. ET is thought tobe
released in hypoxia (5, 6) SQ these effects on veins could be an
important contribution to the overall reactions of the body to
We thank Ms. J. Mitchell and Mr. P. Lidbury for technical
assistance and Dr. R. Botting for editorial help. E.A. thanks
Fundagdode Amparo a Pesquisa do Estado de Sdo Paulo, Brazil
(87/2381-5). P.D.J. is a Fellow of the Canadian Heart Foundation.
The William Harvey Research Institute is supported by a grant from
Glaxo Group Research, Ltd.
Moncada, S., Gryglewski, R., Bunting, S. & Vane, J. R. (1976)
Nature (London) 263, 663-665.
Furchgott, R. F. & Zawadzki, J. V. (1980) Nature (London)
Palmer, R. M. J., Ferridge, A. G. & Moncada, S. (1987)
Nature (London) 327, 524-526.
DeMey, J. G. & Vanhoutte, P. M. (1982) Circ. Res. 51, 439-
Rubanyi, G. M. & Vanhoutte, P. M. (1985) J. Physiol. (Lon-
don) 364, 45-56.
Holden, W. E. & McCall, E. (1984) Exp. Lung Res. 7, 101-112.
Harder, D. R. (1987) Circ. Res. 60, 102-107.
Katusic, Z. S., Shepherd, J. T. & Vanhoutte, P. M. (1987)Am.
J. Physiol. 252, H671-H673.
Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y.,
Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. & Masaki, T.
(1988) Nature (London) -332, 411-415.
Vane, J. R. (1964) Br. J. Pharmacol. Chemother. 23, 360-373.
Piper, P. J. & Vane, J. R. (1969) Nature (London) 223, 29-35.
Salmon, J. A. (1978) Prostaglandins 15, 383-397.
Ferreira, S. H. & Vane, J. R. (1967) Br. J. Pharmacol. Che-
mother. 30, 417-424.
McGregor, D. D. (1965) J. Physiol. (London) 177, 21-30.
Warner, T. D., de Nucci, G. & Vane, J. R. (1988) Br. J.
Pharmacol. 94, 335P.
Cocks, T. M., Angus, J. A., Campbell, J. H. & Campbell,
G. D. (1985) J. Cell. Physiol. 123, 310-320.
Gryglewski, R. J., Moncada, S. & Palmer, R. M. J. (1986) Br.
J. Pharmacol. 87, 685-694.
de Nucci, G., Gryglewski, R. G., Warner, T. D. & Vane, J. R.
(1988) Proc. Nati. Acad. Sci. USA 85, 2334-2338.
de Nucci, G., Warner, T. D. & Vane, J. R. (1988) Br. J.
Pharmacol., in press.
Antunes, E., de Nucci, G. & Vane, J. R. (1988) J. Physiol.
(London), in press.
Warner, T. D., de Nucci, G. & Vane, J. R. (1988) Br. J.
Pharmacol., in press.
Schramm, M., Thomas, G., Towart, R. & Franckowiak, G.
(1983) Nature (London) 303, 535-537.
Bakhle, Y. S. (1980) Eur. J. Pharmacol. 68, 493-496.
Proc. Natl. Acad. Sci. USA 85(1988)