Endothelin-1 causes systemic vasodilatation in anaesthetised turtles (Trachemys scripta) through activation of ETB-receptors
1 Department of Zoophysiology, Institute of Biological Sciences, University
of Aarhus, Denmark
2 Department of Molecular Pharmacology, Physiology and Biotechnology, Brown
University, Providence, RI 02912, USA
* Author for correspondence (e-mail: nini.jensen{at}biology.au.dk)
Accepted 15 August 2005
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Summary |
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Key words: turtle, Trachemys, reptile, blood flow, blood pressure, systemic circulation, pulmonary circulation, endothelin, ETA-receptor, ETB-receptor
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Introduction |
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ET-1 is regarded as one of the most potent vasoconstrictors in mammals
(Yanagisawa et al., 1988,
1989), and ET-1 also exerts cardiovascular effects in ectothermic vertebrates
(Olson et al., 1991
;
Poder et al., 1991
; Wang et
al., 1999
,
2000
;
Hoagland et al., 2000
;
Platzack et al., 2002
). In
fish and mammals, infusion of ET-1 normally gives rise to an initial
vasodilatation that is followed by a long-lasting vasoconstriction
(Yanagisawa, 1989; Olson et al.,
1991
). However, in the alligator, the initial vasodilatation is
very pronounced and long-lasting, whereas the subsequent constriction is small
and only prevalent after large dosages
(Platzack et al., 2002
). Thus,
it seems that the cardiovascular actions of ET-1 differ markedly between
reptiles and mammals (Platzack et al.,
2002
). Reptiles, however, are a phylogenetically very diverse
group with large differences in cardiovascular structure and function
(Page, 2000
). More studies on
other groups of reptiles are, therefore, required to reveal whether the
differences between alligators and mammals apply to all reptiles. ET-1 has
been shown to cause a marked contraction of isolated vascular rings of
pulmonary and systemic arches from turtles
(Poder et al., 1991
). These
vessels, however, contribute little to vascular resistances.
ET-1 is primarily expressed and released from the vascular endothelium and
acts in a paracrine fashion on ETA- and ETB-receptors
that are located on the vascular smooth muscle and the endothelium
(Yanagisawa, 1989; Miyauchi and Masaka,
1999; Masaki,
2004
). In mammals, stimulation of ETA-receptors,
normally located within the smooth muscle, causes constriction. Stimulation of
ETB-receptors within the endothelium leads to vasodilatation,
through the release of nitric oxide, whereas stimulation of
ETB-receptors within the smooth muscle causes constriction
(Mateo and Artiñano,
1997
; Masaki,
2004
). In mammals, the initial dilator response to ET-1 is
normally ascribed to stimulation of ETB-receptors whereas the
pressor response is ascribed to stimulation of ETA-receptors. The
role of these two different receptors has not been studied in reptiles, but
ETB-receptors have been located in various tissues of lizards
(De Falco et al., 2002
).
In the present study, we describe the effects of ET-1 on the systemic and
pulmonary vasculature in anaesthetised turtles (Trachemys scripta).
The cardiovascular system of this turtle is one of the most well-studied
amongst reptiles (Hicks et al., 1998), but very little is known about the role
of endothelium-derived factors (see
Crossley et al., 2000). Through
infusion of a selective ETB-agonist, ETB- and
ETA-specific antagonists and the general ET-antagonist tezosentan,
we also assess which of the two receptor types is responsible for the
haemodynamic changes caused by ET-1.
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Materials and methods |
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A bone saw was used to expose the central blood vessels by removing a
5x5 cm piece of the plastron. The left carotid artery was occlusively
cannulated with a PE50 catheter filled with heparinised saline, while the left
pulmonary artery was non-occlusively cannulated with an intravenous catheter
(Terumo Surflo, Leuven, Belgium) using the Seldinger technique
(White et al., 1989). All
catheters were connected to Baxter Edward (model PX600; Irvine, CA, USA)
disposable pressure transducers, and the signals were amplified using an
in-house-built preamplifier.
For measurements of blood flows, 2S or 2R transit-time ultrasonic blood flow probes (Transonic System, Inc., Ithaca, NY, USA) were placed around the left aortic arch (LAo) and the left pulmonary artery (LPA). Acoustical gel was infused around the blood flow probes to enhance the signal. Both flow probes were connected to a Transonic dual-channel blood flow meter (T206). Signals from the pressure transducers and the blood flow meter were recorded with a Biopac MP100 data acquisition system (Biopac Systems, Inc., Goleta, CA, USA) at 100 Hz.
Experimental protocols
The study consisted of two separate experimental protocols on different
animals. All experiments were carried out at room temperature
(2022°C).
Effects of increased dosages of ET-1 and the effects of ETB-agonist and ETA-antagonist (Protocol 1)
Haemodynamic variables were allowed to stabilise over a period of 45 min
after instrumentation so that baseline values could be obtained. Then, 1 ml
kg-1 of isotonic saline (0.9% w/v) containing 0.1% (w/v) albumin
was given as a sham infusion to evaluate whether the vehicle for ET-1 had
haemodynamic effects. Eight animals then received a series of bolus injections
of increasing doses of ET-1 as follows: 0.4, 1.2, 4, 12, 40, 120 and 400 pmol
kg-1. To perform a preliminary investigation into the ET-receptor
subtypes involved in the haemodynamic changes observed during the
doseresponse characterisation, we then infused, in the following
sequence, the ETB-agonist BQ-3020 (150 pmol kg-1; 0.15
µmol l-1), ET-1 (120 pmol kg-1; 0.1 µmol
l-1), the ETA-antagonist BQ-610 (0.15 µmol
kg-1; 0.15 mmol l-1) and finally ET-1 (120 pmol
kg-1) in seven of the eight turtles. All drugs were administered
through an arterial catheter in 1 ml kg-1 aliquots. Haemodynamic
variables were allowed to return to baseline between each injection, which
took up to 30 min at the highest dosages, and the ETA-antagonist
was allowed 5 min to distribute and take effect before the subsequent
injection of ET-1. A typical Protocol 1 lasted between 4 and 5 h.
Effects of specific inhibition of ETB-receptors and general block of ET receptors (Protocol 2)
Because the first experimental series revealed a pronounced systemic
vasodilatation and similar effects after infusion of the
ETB-agonist, we characterised the effects of ET-1 before and after
specific blockade of the ETB-receptors with the
ETB-antagonist BQ-788 (0.15 µmol kg-1; 0.15 mmol
l-1; N=5). This was followed by an additional infusion of
ET-1 after administration of the general ET-antagonist tezosentan (15.4
µmol kg-1; 15.4 mmol l-1), which blocks both
ETA- and ETB-receptors
(Clozel et al., 1999).
Tezosentan was obtained as a generous gift from Actelion Pharmaceuticals (Allschwill, Switzerland), whilst all other drugs were purchased from Sigma.
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Data analysis and statistics
All recordings of blood flows and pressures were analysed using
AcqKnowledge data analysis software (version 3.7.1.; Biopac). All data
presented in the figures were evaluated with a one-way analysis of variance
(ANOVA) for repeated measures followed by Tukey or Dunnett post-hoc
tests. Data presented as percentages, which are not normally distributed, were
analysed statistically after an arcsine transformation. The effects of the
agonist and antagonists, which are presented as absolute values in the tables,
were assessed with paired t-tests. Differences were considered
statistically significant at a 95% level of confidence (P<0.05),
and all data are presented as means ± S.E.M.
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Results |
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The effects of subsequent infusion of ET-1, the ETB-agonist and
the ETA-antagonist are listed in
Table 1. The
ETB-agonist elicited haemodynamic changes that were qualitatively
similar to those following ET-1, causing a reduction in
Psys and an increase in
sys and
Gsys. Infusion of the ETA-antagonist resulted
in a small increase in
sys,
but Gsys and Gpul were not affected
(Table 1). Blood flows
decreased by 2030% during Protocol 1 and may reflect progressive
deterioration of the experimental preparation, but may also be caused by a
decrease in sympathetic tone and changes in central blood volume. This
progressive decline in flows did not occur in the shorter experimental
Protocol 2.
Effects of specific inhibition of ETB-receptors and general block of ET receptors
The relative changes in the haemodynamic responses to ET-1 (120 pmol
kg-1) before and after administration of the
ETB-antagonist and tezosentan are shown in
Fig. 4, and the absolute values
are listed in Table 2. The
overall responses to ET-1 were similar to those observed in the first
experimental protocol, with the exception of the increase in
sys being non-significant
(P=0.052). Infusion of the ETB-antagonist had no effects
on vascular conductances but caused a rise in Psys,
pul and
tot
(Table 2). All effects of ET-1,
however, were abolished after ETB-receptor blockade
(Fig. 4). The subsequent
treatment with the general ET-receptor antagonist tezosentan did not produce
effects that differed from the treatment with ETB-antagonist, and
the blockade of ET-1 responses persisted.
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Discussion |
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The lack of a secondary pressor response differs from mammals. In mammals,
ET-1 causes an initial, but transient, vasodilatation that usually lasts for
less than 1 min, which is followed by a prolonged and dose-dependent
vasoconstriction that reaches a plateau 510 min after infusion and
often persists for more than 30 min (Mateo
and Artiñano, 1997). This response occurs in both
anaesthetised and awake rats (Knuepfer et
al., 1989
), so it is unlikely that the differences between mammals
and reptiles are due to the influence of anaesthesia. Trout and alligators
also exhibit a biphasic response (Olson et
al., 1991
; Wang et al.,
2000
; Platzack et al.,
2002
), but the initial vasodilatation in alligators is more
pronounced and longer in duration than that reported for mammals, and the
subsequent systemic vasoconstriction is only present after large dosages of
ET-1 (Platzack et al., 2002
).
The lack of a vasoconstriction in turtles may seem surprising, as Poder et al.
(1991
) showed that rings of
the LAo and the pulmonary artery from Trachemys scripta constrict
in vitro when exposed to ET-1. Similar responses were obtained on
aortic rings from catfish and frogs (Poder
et al., 1991
). It is possible that the endothelium was damaged
during these ring preparations, which would reduce the initial dilatation
resulting from endothelial ETB-receptor stimulation, or that the
constrictor response is confined to these large vessels, which do not
contribute significantly to overall vascular resistance.
An abundant occurrence of ETB-receptors in endothelial cells has
been localised in various tissues of the lizard Podarcis sicula
(De Falco et al., 2002), but
our study is the first to investigate the haemodynamic role of the different
ET-receptors in a non-mammalian tetrapod. Two endothelin receptors have been
cloned and pharmacologically characterised in mammals
(Sokolovsky, 1995
). The
ETA-receptor has high affinity for ET-1 and ET-2 but low affinity
for ET-3, while the ETB-receptor has similar affinities for all
three isoforms. In Xenopus, an ETC-receptor has been
cloned from heart and lungs, which is pharmacologically similar to the
mammalian ETA-receptor, with the exception of being insensitive to
the ETA-antagonist BQ-123
(Kumar et al., 1994
).
Stimulation of ETA-receptors, located within the smooth muscle,
generally causes vasoconstriction, and stimulation of ETB-receptors
located in the endothelium leads to vasodilatation
(Mateo and Artiñano,
1997
; Masaki,
2004
). However, ETB-receptors are also located in
smooth muscle, and stimulation of these receptors can lead to constriction
(Mateo and Artiñano,
1997
). This seems to be the case in isolated aortic rings from
sharks, where stimulation of ETB-receptors leads to constriction in
the presence and absence of an intact endothelium
(Evans et al., 1996
).
In turtles, the ETB-agonist BQ-3020 caused a pronounced
vasodilatation that qualitatively resembled that elicited by ET-1, and the
entire response to ET-1 could be completely abolished by the
ETB-antagonist BQ-788. Subsequent treatment with the general ET
receptor antagonist tezosentan did not produce effects that differed from
those of the ETB-antagonist. These observations clearly indicate
that ETB-receptors mediate the systemic vasodilatation in response
to ET-1 in Trachemys. In mammals, the vasodilatation following
ETB-receptor stimulation is often mediated by increased NO
liberation (Moritoki et al.,
1993). Turtles exhibit the same systemic vasodilatation in
response to exogenously administrated NO as other vertebrates
(Crossley et al., 2000
), but
the possible role of NO in the dilation following ET-1 was not investigated in
our study. However, the initial systemic vasodilatation caused by ET-1 was not
affected by NOS inhibition in alligators, suggesting that
ETB-receptors activate other endothelium-derived relaxing factors,
such as prostaglandins or leukotrienes
(Platzack et al., 2002
).
There are no good and easily available ETA-agonists, so the putative involvement of ETA-receptors was only investigated by blockade of ETA-receptors. While our data do show an attenuated cardiovascular response to ET-1 after treatment with the ETA-antagonist, it is likely that this reduction in the response reflects tachyphylaxis, as there seems to be a progressive reduction in the responsiveness throughout the experimental protocol (Table 1). Also, given that ETA-receptor stimulation normally induces a pressor response in fish and mammals, we would have expected ETA-receptor blockade to enhance the vasodilatation in response to ET-1. Thus, it seems unlikely that ETA-receptors are important in the systemic vasculature of turtles.
There were only small direct effects of the various ET-antagonists used in
our study (Tables 1,
2). This indicates that
endothelin contributes very little to the maintenance of the systemic vascular
tone in anaesthetised turtles but does not rule out the possibility that
endothelin may have more pronounced effects in fully recovered animals under
various conditions. Because turtles exhibit pronounced cardiovascular changes
during the intermittent breathing pattern (Shelton and Burggren, 1979;
White et al., 1989;
Wang and Hicks, 1996
), it is
convenient to study the roles of various local factors in anaesthetised
animals where autonomic regulation of the cardiovascular system is reduced
(Crossley et al., 1998
).
The pulmonary vascular conductance of turtles was not affected by ET-1 or
any of the ET agonists and antagonists. Thus, the reduction in
pul that follows the
infusion of ET-1 reflects the systemic vasodilatation that directs blood flows
from the undivided ventricle towards the systemic circulation. Therefore, ET-1
induced a significant rise in the right-to-left cardiac shunt
(Fig. 3D). The lack of an
effect of ET-1 is consistent with the alligator, where only very high dosages
affected the pulmonary circulation
(Platzack et al., 2002
).
However, ETB-receptors are abundant in the endothelium of the
pulmonary arteries in the lizard Podarcis sicula
(De Falco et al., 2002
), and
isolated rings of the pulmonary artery from turtles constrict in response to
ET-1 (Poder et al., 1991
). In
the mammalian lung, endothelin normally causes vasoconstriction and has been
implicated in pulmonary hypertension as well as hypoxic pulmonary
vasoconstriction (Goldie et al.,
1996a
,b
;
Michael and Markewitz, 1996
).
Interestingly, in fish gills, the functional analogue to the lungs of
tetrapods, ET-1 leads to a marked vasoconstriction that seems to be caused by
contraction of the pillar cells in the gill lamellae
(Sundin and Nilsson, 1998
;
Stensløkken et al.,
1999
). The lack of effects of ET-1 in the turtle lung is
consistent with the very small effects of various regulatory peptides and NO
in the pulmonary circulation of most reptiles studied so far
(Crossley et al., 2000
;
Platzack et al., 2002
; Galli
et al.,
2005a
,b
;
Skovgaard et al., 2005
).
In conclusion, the present study reveals a potentially important role for ET-1 in regulating systemic vascular tone in turtles. However, unlike mammals, but consistent with alligators, the primary role of this endothelium-derived peptide appears to be vasodilatation. In turtles, this response is mediated through stimulation of ETB-receptors, whereas ETA-receptors are of minor importance.
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Acknowledgments |
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