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Laboratoire de Neurophysiologie, Unité de Formation et de Recherche de Médecine, Université de Bretagne Occidentale, 29285 Brest Cedex, and European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U-413, Unité Associée au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The central and peripheral cardiovascular effects of endothelin (ET)-1 and ET-3 were investigated in conscious rainbow trout. Both intracerebroventricular and intra-arterial injections of ET-1 (6.25-25 pmol) but not ET-3 (25 pmol) caused a dose-dependent increase in mean dorsal aortic blood pressure and a concomitant decrease in heart rate. The hypertensive effects induced by intra-arterial and intracerebroventricular injection of ET-1 were associated with a significant (P < 0.05) increase in systemic vascular resistance. Intracerebroventricular injection of ET-1 induced a twofold higher pressor response than that caused by intra-arterial injection of ET-1 and provoked a barostatic gain that was reduced by 2.5- to 3-fold compared with that calculated after intra-arterial administration of the peptide. The ET receptor antagonist bosentan significantly (P < 0.05) attenuated these responses regardless of the route of administration. Finally, intra-arterial injection of ET-1 did not significantly modify plasma cortisol level. The present data demonstrate that intracerebroventricular and intra-arterial administration of very low doses of ET-1 produces hypertension in conscious trout. The lack of effect of ET-3 indicates that the hemodynamic actions of ET-1 are mediated both centrally and peripherally through ETA receptors.
bosentan; arterial blood pressure; heart rate; cardiac output
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE TERM ENDOTHELINS (ETs) designates a family of biologically active peptides initially isolated from the culture medium of porcine endothelial cells (33). The three ETs characterized so far (ET-1, ET-2, and ET-3) exhibit vasoconstrictor activity, ET-1 being the most potent isoform. ET-1 causes contraction of arterial and venous preparations in vitro (33) and exerts a positive inotropic effect on isolated mammalian cardiac tissue (11). Intravenous injection of ET-1 in conscious rats produces a transient depressor effect followed by a sustained and potent pressor response that is associated with a decrease in heart rate (HR) (28).
There is now evidence that ETs act as central regulators of mammalian cardiovascular functions. The occurrence of ETs and ET receptors has been demonstrated in the central nervous system of human and rat (9, 15). In addition, intracerebroventricular or intrathecal administration of ET-1 induces a marked increase in arterial blood pressure generally associated with a decrease in HR in conscious rats (10, 25, 28).
In lower vertebrates, a series of peptides called sarafotoxins that share sequence similarities with ETs have been isolated from the venom of the snake Actractaspis engaddensis (30). Recently, the occurrence of ET-1-like immunoreactivity has been demonstrated in the central and peripheral nervous system of fish (12, 21, 35). The existence of specific binding sites for sarafotoxin-b and ET-1 has been reported in the heart and brain of tilapia and torpedo (35), and the presence of ETA-like receptors has been demonstrated in the gill of the rainbow trout (20). Although immunoreactive ETs have been detected in the plasma of amphibians and fish (32), very few studies have been conducted to investigate the effects of ETs in lower vertebrates. In vitro, ET-1 stimulates corticosterone and aldosterone secretion from the frog adrenal gland (5) and causes constriction of isolated blood vessels in fish (23, 31), amphibians, and reptiles (26). In nonanesthetized fish, intra-arterial injection of ET-1 produces vascular responses that are similar to those observed in mammals (23). However, to date, central cardiovascular effects of ET-1 have not been reported in nonmammalian vertebrates.
The aim of the present study was to investigate the cardiovascular effects of intracerebroventricular and peripheral injections of picomolar doses of ET-1 in the rainbow trout, which has proven to be a very appropriate experimental model to determine the hemodynamic responses to regulatory peptides (18, 19, 23). We have also investigated the mechanisms involved in the cardiovascular activity of ET-1 in conscious trout.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chemicals
Synthetic ET-1 and ET-3 were purchased from Neosystem (Strasbourg, France). Norepinephrine was obtained from Sigma (St. Louis, MO). Bosentan or Ro-47-0203 (4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide sodium salt) was a generous gift from Hoffmann-La Roche (Basel, Switzerland). ET-1 and ET-3 were stored in stock solution (10
4 M) at 
25°C.
Peptides and norepinephrine were diluted in Ringer buffer (vehicle)
just before use. Bosentan was initially dissolved in distilled water
and diluted to the desired concentration with Ringer buffer. The
composition of the Ringer solution was (in mM) 124 NaCl, 3 KCl, 0.75 CaCl2, 1.30 MgSO4, 1.24 KH2PO4,
25 NaHCO3, 10 glucose (pH 7.8).
All solutions were sterilized by filtration through 0.22-µm filters
(Millipore, Molsheim, France) before injection.
Animals
Adult rainbow trout (Oncorhynchus mykiss, 290 ± 3.8 g) were purchased locally and maintained in a cylindrical tank containing 1,000 liters of refrigerated (12 ± 1°C), dechlorinated, and aerated tap water. Fish were maintained under a standard photoperiod (lights on 0900-2000) for at least 8 days before the beginning of the experiments. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.Surgical Procedures
The surgical procedures for dorsal aorta cannulation and intracerebroventricular guide placement have been described previously in detail (19). Briefly, trout were anesthetized by immersion in tricaine methanesulfonate [3-aminobenzoic acid ethyl ester (Sigma); 60 mg/l of tap water], and the dorsal aorta was cannulated with a PE-50 catheter (Clay Adams). A 25-gauge needle fitted with a PE-50 catheter was inserted into the third ventricle of the brain, so that the injection of test substances occurred at the level of the preoptic nuclei. The intracerebroventricular injector was made from a 33-gauge stainless steel cannula connected with a PE-10 tubing to a 10-µl Hamilton syringe. In a few experimental fish (n = 11), the cardiac output (
) was also measured. A midline incision was made
through the skin and muscle immediately anterior to the base of the
pectoral fins at a position overlying the ventral aorta, as previously
described (8). A cuff-type Doppler probe (2.0 mm ID; Iowa Doppler
Products, Iowa City, IA) was placed around the ventral aorta. As
previously reported (8), bleeding was usually minimal during the
operation. The incision was sutured and the leads from the flow probe
were secured to the skin sutures. The trout was allowed to recover from
the anesthesia and placed into an experimental 6-liter aquarium, which
was painted black and supplied with partially recycled and aerated tap
water (11-13°C). Oxygen tension in the water tank (YSI model
57 oxygen meter; 20.0 kPa) and pH (7.50-7.80) were continuously
recorded and maintained at constant levels. A small horizontal aperture
was made along the upper edge of the aquarium for connection of the
dorsal aorta cannula and intracerebroventricular injections of
substances without disturbing the trout.
Experimental Protocols
General controls of homeostasis mechanisms before injections. Operated trout were allowed 24 h to recover from surgery and to be accustomed to their new environment. Each day, small amounts of blood were taken from the dorsal aorta to ascertain that the general homeostasis mechanisms of fish were not impaired. One hundred microliters of blood was taken from the dorsal aorta in heparinized tubes (25 U/100 µl). Blood was centrifuged (12,000 g, 4°C, 15 min), and a 50-µl aliquot of plasma was stored at20°C. Hematocrit was
determined by a microcapillary method (microhematometer, Hawkslay). A
10-µl sample of plasma was also used to determine osmolality (vapor
pressure osmometer, Wescor 5500). Plasma cortisol level was determined
at random on 23 experimental fish during the 10 mo of the experiment
(from September to June). After extraction in absolute ethanol,
cortisol concentration was determined by radioimmunoassay (16). The
sensitivity threshold of the assay was 2.5 pg, and values were
corrected for recovery (70.3%).
Test for cardiovascular normality and vascular reactivity. The pressure pulsatility (ratio of the pulse pressure to the mean blood pressure) was determined before each experimental session and used as an index of cardiovascular normality (19). When the pulsatility was <0.10, the trout was discarded. A single intra-arterial injection of norepinephrine (0.62 nmol) was used to check the usual cardiovascular reactivity to exogenous adrenergic agents. Only trout that displayed an immediate increase in diastolic blood pressure (DP) and systolic blood pressure (SP) of ~5% over baseline and a concomitant fall in HR of ~10% under baseline were selected for further experiments. Over 90% of the experimental animals fulfilled these criteria. Once baseline levels of mean dorsal aortic blood pressure (PDA) and HR were stabilized (~2 h), the experimental session for intracerebroventricular or intra-arterial injections started. For all protocols, fish received a control injection of vehicle and, 30 min later, an injection of peptide. The animals usually received a single injection of peptide per day. When two injections were made, a delay of at least 6 h was observed between the injections to avoid tachyphylaxis.
Intracerebroventricular administration of ET-1 or
ET-3. A total of 39 fish received
intracerebroventricular injection of ET-1 or ET-3. The injector was
preloaded with distilled water. A small bubble was created at the level
of the PE-10 tubing, and the injector was loaded with vehicle or ET
solution. The injector was inserted into the intracerebroventricular
guide and, once the cardiovascular parameters had stabilized, the
recording session started for 30 min. After 5 min of recording
(baseline), 0.5 µl of vehicle (Ringer buffer) or 0.5 µl of ET-1
solution was injected over 30 s into the third ventricle. Pilot
experiments showed that intracerebroventricular injection of ET-1, at
doses >50 pmol, caused rapid death of the animals. Therefore, in the
present study, ET-1 was tested at doses of 6.25, 12.5, and 25 pmol
(equivalent to 
21.5, 43, and 86 pmol/kg, respectively). These
picomolar doses are in the same range as those applied previously for
intracerebroventricular injections in rats (27, 28). They are lower
than the doses generally applied intracerebroventricularly to test the
hypertensive effects of other neuropeptides in fish (18). ET-3 was only
tested at a dose of 25 pmol (equivalent to 86 pmol/kg).
Intracerebroventricular administration of
bosentan. The nonpeptide antagonist of ET receptors
Ro-47-0203 (bosentan) (4) was used to test the specificity of the
PDA and HR responses to intracerebroventricular injections of ET-1. A total of 27 fish received
an injection of 17.5 nmol of bosentan into the third ventricle. In rat,
intracerebroventricular injection of ET receptor antagonists is usually
performed at least 15 min before the administration of ETs (27). Thus,
in the present study, intracerebroventricular administration of ET-1
(6.25, 12.5, and 25 pmol; equivalent to 21.5, 43, and 86 pmol/kg,
respectively) was performed 30 min after the injection of bosentan.
Intra-arterial administration of ET-1 or
ET-3. A total of 31 fish received intra-arterial
injection of ET-1 or ET-3. Five minutes after the beginning of the
recording session, 50 µl of vehicle or ET-1 (6.25, 12.5, and 25 pmol;
equivalent to 21.5, 43, and 86 pmol/kg, respectively) or ET-3
solution (25 pmol; equivalent to 86 pmol/kg) was injected through
the dorsal aorta and immediately flushed with 150 µl of vehicle. To
prevent the recording of the pressure artifact due to the injection,
the computer was stopped for 10 s during intra-arterial injections.
Intra-arterial administration of
bosentan. A total of 19 fish received intra-arterial
injection of 1.75 µmol of bosentan. In rat, intra-arterial injection
of bosentan at the same dose (3 mg/kg) is usually performed 5 min
before the administration of ETs (4). In addition, pilot experiments
showed that intra-arterial injection of bosentan 30 min before the
intra-arterial administration of ET-1 did not modify the cardiovascular
effects evoked by the peptide. So, bosentan was injected through the
intra-arterial cannula 5 min before the intra-arterial administration
of ET-1 (6.25, 12.5, and 25 pmol; equivalent to 21.5, 43, and 86 pmol/kg, respectively).
Measurement of Plasma Cortisol Level
The effect of peripheral administration of ET-1 on cortisol secretion was investigated in 10 trout. A sample of 100 µl of blood was taken just before the recording session and replaced by the same volume of Ringer buffer. The animals then received an intra-arterial injection of ET-1 (25 pmol; equivalent to86 pmol/kg). Samples of blood (100 µl
each) were taken 12.5 and 25 min after the injection of the peptide and
replaced by the same volume of Ringer buffer. Plasma cortisol levels
were measured by radioimmunoassay as described above.
Recording of PDA, HR, and
, and Processing of Data
kHz). The
existence of a linear correlation between the Doppler signal and the
mean blood flow has been previously demonstrated in fish (1). SP, DP,
pulse pressure (pulse pressure = SP DP), HR, and
were processed by a digital oscilloscope (Gould 1604), and the data were transferred every 2 s to a 486 personal computer. The PDA
[PDA = (SP + DP)/2],
the pressure pulsatility (pulsatility = pulse
pressure/PDA), HR,
, and systemic vascular resistance (SVR = PDA/) were
also calculated offline by the computer for the preinjection period
(control period, 0-5 min) and the postinjection period (5-30
min). The mean maximum value of these parameters was determined during
the postinjection period (5-30 min). Central venous blood pressure
was assumed to be zero for the calculation of SVR (13). The barostatic
gain, corresponding to the change in the HR per unit change in
PDA (8), was also calculated for
preinjection and for postinjection maximal values of HR and
PDA. Results for cardiovascular
parameters are expressed either as absolute values (HR in beats/min,
PDA in mmHg), arbitrary units
( in kHz, SVR in mmHg/kHz), or percentages of
the control values.
Statistical Analysis
All data are expressed as the mean (±SE) for 8-24 experiments. Data were analyzed by Student's paired t-test or by one-way analysis of variance with repeated measures or by two-way analysis of variance. When appropriate, the multiple-range test of Student-Newman-Keuls was used subsequently to determine significant differences within and between groups. The criterion for statistical significance was P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preinjection values for hematocrit and plasma osmolality were 24.6 ± 1.5% and 285 ± 1.3 mosmol/kgH2O, respectively. The mean plasma cortisol level was 39.8 ± 3.4 ng/ml. The baseline value of the PDA ranged between 22 and 26 mmHg and the spontaneous HR was 55-68 beats/min.
Effect of Intracerebroventricular Administration of ET-1 or ET-3 on Cardiovascular Parameters
The effect of intracerebroventricular injection of ET-1 (25 pmol) on PDA and HR is illustrated in Fig. 1. The peptide provoked a gradual increase in PDA, which reached a maximum (8.2 ± 1.9 mmHg above baseline level) within 15 min after the end of ET-1 administration (Fig. 1A). Then, PDA remained significantly elevated during the whole recording period. Concurrently, intracerebroventricular injection of ET-1 significantly decreased HR 15-20 min after ET-1 administration (Fig. 1B). Both PDA and HR returned to preinjection values within 2 h after ET-1 administration (data not shown). No mortality was observed after intracerebroventricular injection of 25 pmol of ET-1. At the end of the recording session, some of the animals exhibited tail-flip activity. In contrast, intracerebroventricular injection of ET-3 (25 pmol) or vehicle did not cause any significant effect on PDA or HR (Fig. 1) and did not affect the behavior of the animals.
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Intracerebroventricular injection of graded doses of ET-1 (6.25-25
pmol) induced a dose-related increase in
PDA (Fig.
2A). A
significant decrease in HR occurred only after administration of the
highest dose (25 pmol) of ET-1 (Fig.
2B). The peak values for both
parameters were observed within 5-15 min after the injection, depending on the dose. Intracerebroventricular administration of
bosentan (17.5 nmol), 30 min before the injection of ET-1, significantly attenuated the increase in
PDA induced by an
intracerebroventricular injection of 12.5 and 25 pmol of ET-1 (Fig.
2A). In addition, bosentan
significantly attenuated the bradycardia provoked by ET-1
(F1,74 = 11.37;
P < 0.05). In particular, bosentan
totally abolished the bradycardia evoked by 12.5 pmol of ET-1 (Fig.
2B). It was also noticed that
intracerebroventricular administration of bosentan caused by itself a
rapid and transient increase in PDA that peaked 2 min after
injection of the antagonist (2.35 ± 1 mmHg above baseline;
P < 0.05). In contrast,
intracerebroventricular administration of bosentan did not
significantly affect HR.
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In another set of experiments,
PDA, HR, and
were simultaneously monitored after intracerebroventricular injection
of ET-1 (25 pmol). The hematocrit of the fish equipped with a Doppler probe (24.4 ± 2%) was not significantly different from that of other
experimental animals (27.1 ± 2.5%). Central administration of the
peptide caused a significant (P < 0.05) increase of SVR (63%) but had no significant effect on
(Table 1).
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Effect of Intra-Arterial Administration of ET-1 and ET-3 on Cardiovascular Parameters
Intra-arterial injection of ET-1 (25 pmol) provoked an immediate and sustained increase in PDA (Fig. 3A). The maximum effect was reached within 10 min after the injection of the peptide (3.9 ± 0.6 mmHg above baseline level). Intra-arterial injection of ET-1 also decreased HR 5 min after ET-1 administration and throughout most of the recording (Fig. 3B). Both PDA and HR returned to preinjection values within 90 min after ET-1 administration (data not shown). In contrast, intra-arterial injection of ET-3 (25 pmol) or vehicle did not significantly affect PDA (Fig. 3A) or HR (Fig. 3B).
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Intra-arterial administration of graded doses of ET-1 (6.25-25
pmol) produced a dose-dependent increase in
PDA (Fig.
4A) and a dose-related decrease in HR (Fig.
4B). The peak values for both parameters were observed within 5-15 min after the injection, depending on the dose. A two-way analysis of variance applied to the
data shown in Figs. 2 and 4 demonstrated that intracerebroventricular injection of ET-1 induced a greater increase in
PDA
(F1,76 = 6.76; P < 0.05) than did intra-arterial
injection of the same dose of ET-1. In particular, a significant
twofold greater increase in PDA
was observed after intracerebroventricular administration of ET-1 at
doses of 12.5 and 25 pmol compared with the intra-arterial injection of
the same doses of the peptide. In addition, after administration of
ET-1 at doses of 12.5 and 25 pmol, the barostatic gain was reduced
2.5-3 times compared with that calculated after intra-arterial
injection of the same doses of the peptide (for the 12.5 pmol
dose: 1.18 ± 0.54 vs. 2.97 ± 0.34 beats · min1 · mmHg1,
P < 0.05; for the 25 pmol dose:
0.89 ± 0.16 vs. 2.46 ± 0.45 beats · min1 · mmHg1,
P < 0.05).
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Intra-arterial administration of bosentan (1.75 µmol), 5 min before the injection of ET-1, markedly attenuated the effect of the peptide on PDA (Fig. 4A). The bradycardia induced by intra-arterial injection of 6.25 and 25 pmol of ET-1 was also significantly attenuated after intra-arterial pretreatment of trout with bosentan (Fig. 4B). Intra-arterial administration of bosentan alone induced a biphasic effect on PDA, i.e., an immediate and significant (P < 0.05) hypotensive phase (2.9 ± 1.3 mmHg under baseline), followed by a brief and significant (P < 0.05) hypertensive response (1.9 ± 1.2 mmHg above baseline). In contrast, intra-arterial administration of bosentan did not significantly affect basal HR.
In another set of experiments,
PDA, HR, and
were simultaneously monitored after intra-arterial injection of ET-1
(25 pmol). Peripheral administration of the peptide did not affect
but caused a significant increase in SVR (+39%;
Table 1).
Effect of Intra-Arterial Administration of ET-1 on Plasma Cortisol Concentration
The mean cortisol level in trout plasma before administration of ET-1 was 39.2 ± 5.4 ng/ml. Plasma cortisol concentration was unchanged 12.5 and 25 min after intra-arterial injection of ET-1 (47.8 ± 9.8 and 46.6 ± 9.0 ng/ml, respectively).| |
DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The cardiovascular effects of neuropeptides largely depend on the general physiological status and behavior of the animals under experimentation. The present study was conducted in nonanesthetized trout to avoid the effects of anesthetic agents on cardiovascular functions. The hematocrit and the plasma osmolality levels were within the usual range measured in conscious and catheterized trout (19, 29). Cardiovascular parameters were also in the range of values reported in previous studies on rainbow trout (23); in particular, HR level indicated tonic parasympathetic drive to the heart. This observation suggests that the animals had recovered well from the surgical procedures and were adapted to the new environmental situation (8). In addition, the basal plasma cortisol level of the experimental trout was in the same range as that previously determined in cannulated trout within 1-5 days after surgery (2), indicating that the animals had recovered satisfactorily from the surgical stress.
The present study has demonstrated that intracerebroventricular
injection of very low doses of ET-1 provoked a dose-dependent increase
in PDA. These data provide the
first evidence for a hypertensive effect of centrally administered ETs
in a nonmammalian vertebrate. At the highest dose tested (25 pmol),
ET-1 also induced a significant reduction of HR. Several lines of
evidence indicate that the cardiovascular effects observed after
intracerebroventricular injection of ET-1 are attributable to a direct
central action of the peptide rather than a peripheral action.
1) The existence of a blood-brain
barrier has been demonstrated in fish (24), and several reports
indicate that this blood-brain barrier is impermeable to peptide
diffusion. For instance, intracerebroventricular injection of high
doses of 
- or 
-melanocyte-stimulating hormone (up to 0.67 µg)
in conscious killifish does not elicit any darkening of the skin,
indicating that the peptides do not cross the blood-brain barrier (14). Similarly, intracerebroventricular injection of 500 pmol of
melanin-concentrating hormone to trout does not induce any
pallor of the body surface of the animals up to 2 h after the
injection, whereas intra-arterial administration of the same dose of
the peptide induces marked bleaching of the skin within 10 min
(personal unpublished data). 2)
Intracerebroventricular administration of 12.5 and 25 pmol of ET-1
induced an increase in PDA that
was two times higher than that provoked by intra-arterial injection of
the same doses of the peptide. The fact that intracerebroventricular
injection of ET-1 provoked a barostatic gain that was reduced by 2.5- to 3-fold compared with that calculated after intra-arterial
administration of the peptide suggests that, in trout, ET-1 may act
centrally to blunt baroreceptor-mediated cardioinhibitory reflex. The
delay (5-15 min) observed between the administration of ET-1 and
the maximum effect on PDA in trout
was similar to that previously reported in conscious rat after
intracerebroventricular injection of similar doses of ET-1 (8-66
pmol) (25, 27). Changes in arterial pressure may result from variations
of either or SVR (or both). The use of a miniature
Doppler flow probe for measurement of made it
possible to demonstrate that the hypertensive response evoked by
intracerebroventricular injection of ET-1 could be accounted for mainly
by an increase in SVR.
The minimum effective dose of ET-1 applied centrally in trout compares favorably with the threshold doses reported in rat, i.e., ~8-20 pmol (25, 27, 28). It has been observed that intracerebroventricular injection of higher doses of ET in rat causes rotational behavior and convulsions (25, 28), followed by cardiovascular collapse and death (28). We also found that, in trout, intracerebroventricular administration of doses of ET-1 >50 pmol caused the death of the animals. The lethal effect of ET-1 can be likely ascribed to cerebral ischemia because intracerebroventricular injection of ET-1 causes reduction of cerebral blood flow (27). However, it has been reported that, in rat, the initial pressor response evoked by centrally administered ET-1 (30 pmol) is not a consequence of cerebral ischemia (10), suggesting that the hypertensive response induced by intracerebroventricular injection of ET-1 in trout is not secondary to local cerebral vasoconstriction. Although no mortality was observed after injection of 25 pmol of ET-1, at the end of the recording session the trout exhibited tail-flip activity.
The effects of ETs are mediated by at least three types of G protein-coupled membrane receptors that exhibit differential affinities for the various isoforms of ETs (30): the ETA receptor type possesses a higher affinity for ET-1 and ET-2 than ET-3; the ETB receptor does not discriminate between the three isoforms; and the ETC receptor exhibits high affinity for ET-3. The observation that the hypertensive response to intracerebroventricular administration of ET-1 was inhibited by the mixed ETA/ETB receptor antagonist bosentan (4) indicated that the effect of the peptide was mediated through ETA and/or ETB receptors. Because intracerebroventricular administration of ET-3 did not affect PDA or HR, we conclude that the central effect of ET-1 can be ascribed to activation of an ETA receptor subtype. In fact, competition studies have shown the occurrence of selective ET-1 binding sites (presumably corresponding to ETA receptors) in the fish brain (35). Studies conducted in mammals have shown that the cardiovascular responses evoked by intracerebroventricular administration of ETs are mediated by ETA receptors (27). It thus appears that both the physiological action of central ET-1 on hemodynamic functions and its mode of action have been highly conserved during evolution.
Peripheral administration of picomolar doses of ET-1 provoked a clear
dose-related increase in PDA
associated with a decrease in HR. The threshold dose inducing a
significant increase in PDA is in
the same range as those previously reported for other vasoactive peptides, such as arginine vasotocin (19) and urotensin II (18). In a
previous study, Olson et al. (23) reported that bolus intra-arterial injection of 500 ng/kg body wt (200 pmol/kg body wt) of ET-1 in
trout only produced a transient decrease in
PDA, whereas, at doses of 1,500 and 5,000 ng/kg body wt (600 and 2,000 pmol/kg body wt), ET-1
provoked a triphasic pressor-depressor-pressor response. We now
demonstrate that intra-arterial injection of ET-1, at doses 3-30
times lower than those used by Olson et al. (23), only caused a pressor
effect and a concomitant bradycardia. The observation that the
hypertensive response to ET-1 was accompanied by a reduction of
indicates that the rise in
PDA can be accounted for by
systemic vasoconstriction. Consistent with this notion, in vitro
studies have demonstrated that, in fish, ET-1 causes a dose-dependent
contraction of vascular smooth muscles (23, 31). It has been suggested
that in vivo administration of ET-1 could be responsible for coronary
spasm (7), which may affect cardiac performance. However, ET-1 does not
exert any inotropic or chronotropic effects in trout (23). It thus
appears that the reduction in HR and observed in the
present study can be likely ascribed to activation of cardioinhibitory baroreflexes.
Administration of bosentan 5 min before the intra-arterial injection of ET-1 markedly reduced the pressor and chronotropic responses to ET-1, indicating that the peptide exerted its effect through a classical ETA or ETB receptor. In addition, intra-arterial injection of bosentan caused by itself a consistent depressor/pressor response, suggesting the existence of a physiological endothelinergic tonus regulating the basal PDA in trout. Because intra-arterial injection of the selective ETB agonist ET-3 did not affect blood pressure or HR, it appears that the hypertensive and bradycardic effects of ET-1 are mediated by an ETA receptor subtype. Consistent with these data, an ETA-like receptor has previously been characterized in the gills of the rainbow trout (20). Similarly, in rat, ETA receptors are involved in the hypertensive effect induced by intra-arterial administration of ET-1 (30).
In vitro studies have shown that, in amphibians, subnanomolar concentrations of ET-1 stimulate the secretion of corticosteroids through activation of ETA receptors (3, 5). Assuming that the plasma volume of the trout is ~20 ml/kg body wt (6), a bolus injection of 25 pmol of ET-1 would yield a circulating concentration of the peptide of ~4 nM. In fact, Olson (22) has shown that, in fish, ET is readily extracted from the circulation, suggesting that the plasma level of ET-1 after a single intra-arterial injection of the peptide could be much lower. The present study has demonstrated that intra-arterial administration of 25 pmol of ET-1 does not induce a significant increase in plasma cortisol level, suggesting that, in trout, ETs do not play a significant role in the control of adrenal steroidogenesis. In support of this hypothesis, it has been reported that prolonged infusion of ET-1 does not affect urine flow and electrolyte concentrations in trout (23).
In conclusion, the present study has demonstrated that central and peripheral administration of picomolar doses of ET-1 causes a substantial rise in PDA and a concomitant decrease in HR. Both the central and peripheral effects of ET-1 can be ascribed to activation of ETA receptors.
Perspectives
The results of the present study have shown that in trout the hypertensive action of ET-1 in the periphery is matched by a similar qualitative effect of the peptide in the brain. The high potency of ET-1 in evoking the hypertensive response is indicative that the peptide has a physiological role in cardiovascular regulation in trout. Significant pressor effects were observed after both intra-arterial and intracerebroventricular injection of 12.5 pmol of ET-1. In contrast, in the same system, a dose of 300 pmol of trout galanin was required to elicit significant hypertension following intracerebroventricular injection, and a dose of 150 pmol was required to produce significant hypotension following intra-arterial injection (17). The relevance of the data to trout cardiovascular physiology is also emphasized by the recent preliminary report describing the purification and structural characterization of trout ET (J. M. Conlon, personal communication). A single molecular form of the peptide was identified in an extract of trout kidney whose amino acid sequence was the same as human ET-1 in 17 out of 21 residues. It is significant that a peptide corresponding to human ET-3, which did not exhibit biological activity in the trout under the conditions used in this study, was not present in trout tissues.ET is released by the constitutive rather than the regulated secretory pathway, and it is conceivable that ET may exercise its effects in the periphery by a classical hormonal mechanism. Centrally, ET may have a neuromodulatory or a neurotransmitter function acting on nuclei involved in neuroendocrine or neuronal control of cardiovascular functions. Further studies are clearly required to determine the localization of ET-producing elements as well as the distribution of ET-binding sites and to reveal under which circumstances the endothelinergic system of fish is triggered to participate in cardiovascular homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Martine Clozel (Hoffmann-La Roche, Basel, Switzerland) for the gift of bosentan, René Creach for animal care, and Sabrina Mancel and Martine Simon for typing the manuscript.
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FOOTNOTES |
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This study was supported by grants from the Institut de Synergie des Sciences et de la Santé-Brest, Institut National de la Santé et de la Recherche Médicale U-413, the LARC-Neuroscience network, and the Conseil Régional de Haute-Normandie.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. C. Le Mével, Laboratoire de Neurophysiologie (EA 2217), UFR de Médecine, Université de Bretagne Occidentale, 22 Avenue Camille Desmoulins, 29285 Brest Cedex, France (E-mail: jean-claude.lemevel{at}univ-brest.fr).
Received 14 September 1998; accepted in final form 15 December 1998.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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), 25 pmol of endothelin (ET)-1 (
), or 25 pmol of ET-3 (
) on
mean dorsal aortic blood pressure
(PDA;
A) and heart rate (HR;
B) in conscious trout. Arrowhead
indicates onset of injection. Each curve represents mean (±SE at
selected times) of individual recordings from 10-12 fish. bpm,
Beats/min. * P < 0.05 vs.
baseline values (0 and 5 min time points) or vehicle at given time.
