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Vol. 280, Issue 1, 6-15, 1997
Centre de recherche (Université Laval), Hôtel-Dieu de Québec, Québec (Québec) Canada G1R 2J6
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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We investigated the mechanism of the hypotensive effect of Sar-[D-Phe8]des-Arg9-bradykinin (BK) in lipopolysaccharide-treated anesthetized rabbits. The study involved pharmacokinetic and hemodynamic measurements and tests of antagonism with various drugs. The rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma was slower than that of Lys-BK, a naturally occurring B1 agonist. The amplitude of the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK was not affected by pretreatment with indomethacin, diclofenac, dazmegrel, NG-nitro-L-arginine, glibenclamide, MK-886, BN-50739, atropine or propranolol, but its duration was shortened by indomethacin and diclofenac. Sar-[D-Phe8]des-Arg9-BK-induced hypotension was associated with decreases of total peripheral resistance, cardiac output, carotid, mesenteric and femoral blood flow, transient reductions followed by secondary increases of vascular resistance in the carotid and femoral beds, reductions of central venous pressure, but no change of hematocrit. Animal pretreatment with diclofenac or hexamethonium abolished the secondary increases of carotid bed vascular resistance caused by the B1 agonist. These and other results suggest that peripheral vasodilation leading to a decrease of total peripheral resistance and a decrease of cardiac output may both contribute consecutively to the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK in this animal model. Inappropriate compensatory responses to arterial hypotension, prostaglandin release, and slow rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma, may all be at the basis of the prolonged duration of the hypotension caused by the B1 agonist.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The existence of a distinct kinin
B1 receptor regulated by cytokines has been confirmed
recently by the cloning and sequencing of a G protein-coupled receptor
from human cells that is selectively stimulated by
Lys-des-Arg9-BK and blocked by
Lys-[Leu8]des-Arg9-BK (Menke et
al., 1994
). Constitutive B1- and B2-kinin
receptors mediate the cardiovascular effects of kinins in
vivo in the dog (Lortie et al., 1992; Nakhostine
et al., 1993) and cat (DeWitt et al., 1994),
whereas in other species such as the rabbit (Regoli et al.,
1981; Drapeau et al., 1991), swine (Siebeck et
al., 1989) and rat (Tokumasu et al., 1995), the
cardiovascular effects of classical agonists of kinin B1
receptors (i.e., des-Arg9-BK and
Lys-des-Arg9-BK) are mediated by LPS- or cytokine-inducible
kinin B1 receptors, and those of BK itself, by constitutive
kinin B2 receptors (Marceau, 1995).
The sensitization of the cardiovascular system of rabbits to
B1 agonists by LPS, or by components of the cytokine
network (e.g., interleukin-1) (Bouthillier et
al., 1987; deBlois et al., 1991), is currently believed
to result from an up-regulation of kinin B1 receptors
(deBlois et al., 1991; Marceau, 1995). Recent in
vitro binding experiments with vascular smooth muscle cells of
rabbits strongly support the hypothesis that induction of
B1 receptors is responsible for the sensitization of the
cardiovascular system of rabbits treated with LPS or cytokines to
B1 agonists (Schneck et al., 1994; Galizzi
et al., 1994; Levesque et al., 1995). Hypotensive
responses of LPS-treated rabbits to B1 agonists are
inhibited by sequence-related antagonists of B1 receptors (Regoli et al., 1981; Drapeau et al., 1993), but
not by HOE 140, a kinin B2 receptor antagonist (Drapeau
et al., 1993), thus indicating that kinin B1
rather than B2 receptors mediate the hypotensive effect of
B1 agonists in LPS-treated rabbits.
The precise mechanism underlying B1 agonist-induced
hypotension in LPS-treated rabbits is unknown. B1 agonists
are known to relax isolated mesenteric and celiac arteries of rabbits
by a PG-mediated process (Churchill and Ward, 1986; deBlois and
Marceau, 1987; Ritter et al., 1989). They also increase the
production of prostacyclin from isolated rabbit aorta rings and
cultured smooth muscle cells derived from the rabbit aorta (Levesque
et al., 1993). However the hypotensive effect of
des-Arg9-BK, a prototypical B1 agonist, in
LPS-treated rabbits is not reduced by indomethacin treatment (Regoli
et al., 1981). On the other hand indomethacin was shown
recently to reduce the duration, but not the amplitude of hypotensive
episodes elicited by
Sar-[D-Phe8]des-Arg9-BK, a
metabolically protected B1 agonist, in LPS-treated rabbits (Drapeau et al., 1991).
Des-Arg9-BK, acting through B1 receptors, was
shown previously to relax isolated rabbit carotid artery rings (Pruneau
and Bélichard, 1993), an effect which was inhibited by
endothelium removal and N
-nitro-L-arginine.
This result led the authors to suggest that kinin B1
receptors are coupled to the release of endothelium-derived NO. However
the participation of NO in the vasorelaxant effect of B1
agonists in rabbit isolated vascular tissues may be limited to selected
blood vessels, because B1 agonist-induced relaxation of
rabbit celiac artery rings is not dependent on an intact endothelium (Ritter et al., 1989). Whether or not NO mediates part of
the hypotensive effect of B1 agonists in LPS-treated
rabbits is as yet unknown.
The present study investigated the mechanism by which B1
agonists elicit hypotension in LPS-treated rabbits, with
Sar-[D-Phe8]des-Arg9-BK, a
metabolically protected B1 agonist (Drapeau et
al., 1991, 1993; Davis and Perkins, 1994a) as prototype, and a
variety of experimental approaches (e.g., pharmacokinetic
and hemodynamic measurements and inhibitor studies). This study is the
first to describe in detail the cardiovascular consequences of a
persistent stimulation of B1 receptors for kinins. Its
relevance relied on two important points: 1) plasma immunoreactive
des-Arg9-BK is increased in animals pretreated with LPS
(Raymond et al., 1995); 2) B1 receptors for
kinins are up-regulated in LPS-treated animals (see above). Knowledge
from such a study might provide a rationale to decide whether the
inhibition or stimulation of B1 receptors for kinins is
likely to be beneficial to the septic subjects. The results are
consistent with the idea that the hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK in
LPS-treated rabbits involves an early episode of peripheral vasodilation leading to a decrease of TPR, followed by an episode of a
decrease of CO.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Studies were performed with New Zealand White rabbits (1.5-2.0 kg) of either sex. Animals were given free access to standard rabbit chow and tap water, and kept under constant environmental conditions for a few days before use. Experiments were conducted in accordance with the principles and guidelines of the Canadian Council on Animal Care and approved by the local Institutional Committee on Animal Care.
Pharmacokinetic measurements.
The plasma clearance and
biological half-life in plasma (T1/2) of
Sar-[D-Phe8]des-Arg9-BK, a
metabolically protected agonist of B1 receptors for kinins, and of Lys-des-Arg9-BK, a naturally occurring
B1 agonist (Drapeau et al., 1991), were
determined and compared with intact (i.e., no pretreatment with LPS) pentobarbital-anesthetized (see below) rabbits. After implantation of a polypropylene catheter (PE-90) into the left carotid
artery down to the aorta, animals were given an intraarterial injection
of either of the two peptides (3 mg/animal). Arterial blood samples
(2 × 1.35 ml) were collected 1 min before (t = 0) and at different times (1, 3, 5, and 10 min) after injection of the
B1 agonists in 1.5-ml test tubes containing
1,10-phenanthroline and amastatin (final concentrations, 5 mM and 5 µM, respectively, to prevent the enzymatic degradation of the two
B1 agonists), and were centrifuged at 10,000 × g for 1.5 min. Recovered plasmas (0.75 ml) were supplemented
with 4.5 ml of cold (
80°C) ethanol (99% v/v), mixed thoroughly and
allowed to rest for 60 min on ice before being centrifuged again at
2000 × g (at 4°C) for 15 min. Supernatants were
transferred into polypropylene tubes and evaporated overnight in a
Speed-Vac apparatus; the plasma residues were stored at 20°C for 2 to 4 weeks before being analyzed for their content in B1
agonists (see above) by HPLC.
). The distribution phase was assumed to be complete at the
1-min time point after injection.
Effect of various inhibitors on the hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK.
The experiments were aimed at assessing the potential involvement of
secondary mediators in the hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK in the
LPS-treated rabbit. Animals were pretreated with a sublethal dose of
LPS (30 µg/kg) injected into a marginal ear vein 5 h before
anesthesia with sodium pentobarbital (30-40 mg/kg i.v., adjusted
individually). Lidocaine 2% was applied topically at sites of
incision. The trachea was intubated and ventilatory assistance was
provided with a Harvard respiratory pump. The body temperature of
animals was monitored continuously with a rectal thermoprobe connected
to a telethermometer (Yellow Springs Instruments, Yellow Springs, OH),
and kept constant at 38-39°C using a heating pad placed underneath
the animal. A polyethylene catheter (PE-90) was inserted into the left
carotid artery and pushed into the aorta for direct recording of BP
with a pressure transducer (Spectramed P23 XL) connected to a Grass
polygraph (model 79); a three-way valve connector was attached to the
catheter to interrupt the BP recording and to inject drugs
intraarterially. Hypotensive responses to
Sar-[D-Phe8]des-Arg9-BK (750 ng/kg i.a.) were measured in animals pretreated with various inhibitors
(one inhibitor at a time) (see below) and compared with those measured
in drug vehicle-pretreated (control) animals. The inhibitors tested
were (dose in mg/kg): indomethacin (5), diclofenac (5),
NG-nitro-L-arginine (30), dazmegrel (1),
glibenclamide (15), MK-886 (3), BN-50739 (10), atropine (5) and
propranolol (5). The dose of indomethacin selected was previously shown
to acutely suppress the increase of immunoreactive circulating
prostanoids (PG, thromboxanes) induced by the anaphylatoxin C5a in the
anesthetized rabbit (Lundberg et al., 1987). Diclofenac
(also a cyclooxygenase inhibitor) was used at the same dose as
indomethacin for the purpose of comparison. The chosen dose of
NG-nitro-L-arginine, an inhibitor of NO
synthases (Moore et al., 1990), was selected on the basis of
its ability to reduce the NO-mediated hypotensive responses to
cumulative i.a. infusions of ACH (1, 3, 6 and 12 µg/kg/min) in our
animal model. Control animals in these studies had their basal MABP
first increased to levels similar to those of
NG-nitro-L-arginine-treated animals with
phenylephrine (10-50 µg/kg/min i.v.) as vasopressor drug before ACH
infusions (Rees et al., 1989, 1990). Dazmegrel, a potent and
selective thromboxane synthetase inhibitor, was used at a dose
previously shown to reduce serum thromboxane B2 levels by
88% in anesthetized rabbits (Parry et al., 1982).
Glibenclamide, an inhibitor of ATP-sensitive K+ channels
(Edwards and Weston, 1993), was used at a dose shown in preliminary
experiments to reduce by 75% the hypotensive effect of cromakalin (100 µg/kg i.a.), an activator of ATP-sensitive K+ channel
(Edwards and Weston, 1993). The selected dose of MK-886 was previously
shown to be effective as inhibitor of leukotriene biosynthesis in
various animal models (Gillard et al., 1989). BN-50739, an
inhibitor of PAF (Yue et al., 1990), was used at a dose
found in preliminary experiments to suppress the hypotensive effect of
PAF (500 pmol/kg i.a.) in our animal model. Atropine and propranolol
were both used at doses shown in preliminary experiments to suppress
the hypotensive effect of ACH (3 µg/kg i.a.) and isoproterenol (1 µg/kg i.a.), respectively. Each inhibitor (or its vehicle) was
injected intravenously 15 min before injection of
Sar-[D-Phe8]des-Arg9-BK.
Hemodynamic effects of
Sar-[D-Phe8]des-Arg9-BK.
These experiments were performed to obtain further insight into the
mechanism of
Sar-[D-Phe8]des-Arg9-BK-induced
hypotension in LPS-pretreated rabbits. With use of pentobarbital-anesthetized animals we first examined the effect of
bolus injections of
Sar-[D-Phe8]des-Arg9-BK on the
following hemodynamic parameters: MABP, HR, CBF and CVR. In some of
these animals the effect of the B1 agonist on the CVP and
HTC was also monitored. BP was measured as described above. HR was
derived from the BP signal by use of a Grass tachograph (model 7P 44).
CBF was measured with a precalibrated blood flow probe (1.5 mm in
internal diameter) (Skalar Medical, Delft, The Netherlands) placed
around the right common carotid artery and connected to a Skalar
electromagnetic blood flowmeter (model MDL 1401). The flow probe was
zeroed in saline at room temperature before placement on the carotid
artery and the zero was checked in vivo by transient
(~1-2 sec) clamping of the carotid artery upstream of the carotid
flow probe. CBF was recorded on a Harvard recording system (model
52-9545). CVR was calculated by dividing MABP values by CBF values.
CVP measurements were made by a PE-90 catheter introduced into the
right jugular vein and positioned close to the right atrium of the
heart. Pressure transducers (for MABP or CVP) were positioned at the
level of the heart. All animals received 400 U of heparin i.v., and a
30-min period was allowed for the stabilization of hemodynamic
parameters before injection of
Sar-[D-Phe8]des-Arg9-BK (750 ng/kg i.a.). MABP, HR, CBF, CVR, CVP and HTC (via carotid blood samplings) values were measured before (preinjection, control values) (t = 1 min) and different times (0.2, 0.5, 1, 2, 5 or 10 min) after injection of the B1 agonist.
Preliminary studies having shown that
Sar-[D-Phe8]des-Arg9-BK, despite
eliciting relatively prolonged hypotensive episodes in this animal
model, decreases CVR values only transiently, we also examined the
possibility that compensatory mechanisms involving either the
prostanoids and/or the autonomic nervous system contribute to mask the
vasodilatory property of the B1 agonist and its effect on
CVR values. To test this possibility we measured the hemodynamic effects of
Sar-[D-Phe8]des-Arg9-BK in
animals pretreated with the cyclooxygenase inhibitor, diclofenac, or
with the ganglion-blocking drug, hexamethonium. MABP, HR, CBF and CVR
values were measured before (t = 1 min) and at
selected times (0.2, 0.5, 1, 2, 5 and 10 min) after injection of
Sar-[D-Phe8]des-Arg9-BK (750 ng/kg i.a.). The B1 agonist was injected 15 min after animal pretreatment with diclofenac (5 mg/kg i.v.) or hexamethonium (10 mg/kg i.a.).
1 min) and at
selected times (0.2, 0.5, 1, 2, 5 and 10 min) after injection of
Sar-[D-Phe8]des-Arg9-BK (750 ng/kg i.a.). In some experiments,
Sar-[D-Phe8]des-Arg9-BK was
injected in animals in which the basal MVR was artificially raised, to
determine if this condition alters the effect of the B1
agonist on MVR. The increase of basal MVR was obtained by prior i.v.
infusion of animals with the sympathomimetic drug, methoxamine (50-150
µg/min). The B1 agonist was injected 15 min after
starting the methoxamine infusion.
Finally, based on the results of regional blood flow and vascular
resistance studies, we investigated the effect of
Sar-[D-Phe8]des-Arg9-BK on the CO
and TPR of LPS-treated rabbits. These experiments were performed in
pentobarbital-anesthetized rabbits premedicated 30 min before
anesthesia with diazepam (5 mg/kg i.m.). Animals were instrumented for
MABP and HR measurements, as well as for i.a. injections of
Sar-[D-Phe8]-des-Arg9-BK (750 ng/kg), as described above. All animals were ventilated (open-circuit)
with 100% O2 instead of room air, and were given an i.v.
infusion of saline (total volume, 25-50 ml) during the surgical period
only. The thoracic cavity was opened through a midline longitudinal
incision, and the pericardium retracted with small forceps. After
careful dissection of the aortic arch, the blood flow probe (4.5 mm in
internal diameter) was placed around it and used to measure CO. MABP,
HR, CO and TPR were measured before and at different times after
injection of the B1 agonist (as above). TPR was derived
from the ratio MABP/CO.
Drugs.
LPS, extracted from Escherichia coli
serotype O111:B4, was from Difco (Detroit, MI).
Lys-des-Arg9-BK and BK were purchased from Peninsula
Laboratories (Belmont, CA).
Sar-[D-Phe8]des-Arg9-BK was
synthetized in our laboratory by solid-phase methodology (Drapeau and
Regoli, 1988). Halothane was from M.T.C. Pharmaceuticals, Cambridge,
Ontario, and diazepam injectable emulsion from KabiVitrum, Newmarket,
Ontario. Methoxamine (Vasoxyl) was from Burroughs Wellcome (Kirkland, Quebec). Indomethacin, sodium diclofenac,
NG-nitro-L-arginine, glibenclamide, atropine
sulfate, hexamethonium bromide, propranolol hydrochloride,
NG-nitro-L-arginine methyl ester, acetylcholine
chloride, isoproterenol hydrochloride, phenylephrine hydrochloride and
L-
-phosphatidylcholine 
-acetyl-
-O-alkyl (PAF) were
all from Sigma Chemical Co. (St. Louis, MO). Dazmegrel was a gift
from Pfizer Inc., (Groton, CT). MK-886 (or
3-[1-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2-2-dimethylpropanoic acid) was provided by Merck Frost (Pointe-Claire, Dorval,
Québec). BN-50739 (or
6-(2-chlorophenyl)-9-[3,4-dimethoxyphenylthiomethyl) thiocarbonyl]-1-methyl-7,8,9,10-tetrahydro-4H-pyrido[4
,3-4,5]-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine) was a gift from Dr. Pierre Braquet (Institut Henri Beaufour, Le Plessis
Robinson, France). Indomethacin and
NG-nitro-L-arginine were dissolved with 0.1 M
Na2CO3. Dazmegrel was dissolved with 35% (v/v)
ethanol in saline. Glibenclamide was dissolved with 100% DMSO.
Diclofenac was dissolved with 25% DMSO in 0.1 M
Na2CO3. MK-886 was dissolved with 70% (v/v)
ethanol in distilled water. BN-50739 was dissolved with 100% DMSO. All other drugs were dissolved with saline. All drug solutions were made
fresh daily.
Statistics.
Data are mean ± S.E.M. Statistical
analysis was made by the Student's t test for unpaired data
(for results, see fig. 1), or by the Kruskal-Wallis test followed by
the Mann-Whitney test (for other results). P 
.05 was considered
significant.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Figure 1 shows the plasma concentrations of Sar-[D-Phe8]des-Arg9-BK and of Lys-des-Arg9-BK in normal (i.e., no pretreatment with LPS), pentobarbital-anesthetized rabbits at various times after an i.a. bolus injection of 3 mg/animal of either one of these peptides. When plotted with a logarithmic ordinate scale, the same data gave straight lines. The correlation coefficients (r) of the regression lines were .99 in both cases. Application of equations of first-order drug disposition processes to the data provided T1/2 values of 110 ± 10 and 32 ± 6 sec (n = 3 for each peptide) (P < .05) for Sar-[D-Phe8]des-Arg9-BK and Lys-des-Arg9-BK, respectively. Calculated plasma clearance values of both peptides were (in the same order) 54 ± 6 and 162 ± 74 ml/min.
Effect of various inhibitors on the hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK.
Hypotensive effects of
Sar-[D-Phe8]des-Arg9-BK (750 ng/kg i.a.) were measured in LPS-treated, anesthetized rabbits acutely
pretreated with either indomethacin (5 mg/kg), diclofenac (5 mg/kg),
NG-nitro-L-arginine (30 mg/kg), dazmegrel (1 mg/kg), glibenclamide (15 mg/kg), MK-886 (3 mg/kg), BN-50739 (10 mg/kg), atropine (5 mg/kg) or propranolol (5 mg/kg), and compared with
those measured in drug vehicle-treated (control) animals. None of these
inhibitors reduced the amplitudes of hypotensive responses to the
B1 agonist. Thus, decreases in MABP elicited by the
B1 agonist after pretreatment with indomethacin,
diclofenac, NG-nitro-L-arginine, dazmegrel,
glibenclamide, MK-886, BN-50739, atropine or propranolol were (in mm
Hg): 29 ± 3, 36 ± 4, 33 ± 7, 35 ± 3, 38 ± 9, 47 ± 5, 49 ± 5, 41 ± 4 and
53 ± 9, respectively (from base lines of 78 ± 7, 104 ± 5, 102 ± 11, 91 ± 6, 109 ± 6, 125 ± 6, 99 ± 4, 89 ± 3 and 111 ± 3 mm Hg, respectively; n = 4-7). Decreases in MABP in corresponding drug
vehicle-treated (control) animals were (in mm Hg): 30 ± 2, 35 ± 5, 43 ± 8, 46 ± 5, 52 ± 2, 51 ± 11, 47 ± 5, 55 ± 6 and 52 ± 3 (from base lines of 82 ± 3, 89 ± 12, 84 ± 2, 105 ± 4, 109 ± 4, 116 ± 6, 94 ± 8, 98 ± 6 and
97 ± 5 mm Hg; n = 4-7). The times for
half-recovery of hypotensive episodes (TR50) caused by
Sar-[D-Phe8]des-Arg9-BK were 214 ± 43 sec (n = 7) and 71 ± 31 sec (n = 4) (P < .05 when compared with controls) in animals pretreated
with indomethacin or diclofenac, respectively. Corresponding values in
drug vehicle-treated (control) animals were 359 ± 33 sec
(n = 7) and 624 ± 150 sec (n = 4). None of the other drugs affected the TR50 values of the B1 agonist. Hypotensive responses to ACH (1, 3, 6 and 12 µg/kg/min i.a.) (see "Methods") in four animals pretreated with
the NG-nitro-L-arginine solvent (control) were
14 ± 4, 29 ± 6, 47 ± 6 and 55 ± 4 mmHg,
respectively. Corresponding responses in four animals pretreated with
NG-nitro-L-arginine (30 mg/kg) were 7 ± 2 (P > .05), 11 ± 3 (P < .05), 18 ± 3 (P < .005) and 26 ± 2 mmHg (P < .001). Basal MABP
in these animal groups were similar (control, 110 ± 3 mm Hg;
NG-nitro-L-arginine treatment, 104 ± 7 mm
Hg) (P > .05).
Hemodynamic effects of
Sar-[D-Phe8]des-Arg9-BK.
Figure 2 illustrates the time-related hemodynamic
effects of
Sar-[D-Phe8]des-Arg9-BK (750 ng/kg i.a.) in LPS-treated, pentobarbital-anesthetized rabbits. Within
seconds after injection of the B1 agonist the MABP fell
(40 ± 3 mm Hg) and remained lower than the preinjection value
for the next 5 to 10 min. Over the same period the HR decreased gradually to reach its lowest value (30 ± 7 bpm) 5 min after injection of
Sar-[D-Phe8]des-Arg9-BK.
Hypotensive episodes caused by the B1 agonist were
associated with concomitant reduction of CBF, peak CBF reductions being
recorded 1 min after injection of
Sar-[D-Phe8]des-Arg9-BK. The CVR
fell markedly soon (12 sec) after injection of the B1
agonist, returned to the preinjection value at the next time point (30 sec) despite persisting hypotension and later increased significantly
above preinjection values. The CVP exhibited a decrease (1.6 ± 0.6 mm Hg), whereas the HTC remained stable.
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MABP values at the 0.2, 0.5, 1, 2, 5 and 10 min time points after
injection of the B1 agonist were 25 ± 3, 30 ± 4, 34 ± 3, 33 ± 3, 23 ± 4 and 12 ± 3 mm Hg (P < .01 at all time points). HR values at the same time
points were: +3 ± 3, 1 ± 3, 9 ± 5, 17 ± 6, 22 ± 8 and 20 ± 8 bpm (P < .05 at the 1-min
time point and on). Corresponding FBF values were 0 ± 0, 1 ± 0, 4 ± 1, 7 ± 1, 8 ± 1 and 6 ± 2 ml/min (P < .01 at the 1-min time point and on).
Corresponding FVR values were 1.6 ± 0.3, 2.0 ± 0.3, 1.0 ± 0.4, +1.2 ± 0.9, +4.2 ± 1.1 and +4.0 ± 1.0 mmHg/ml/min (P < .01 at all time points, except 2 min where
P > .05).
LPS-pretreated, halothane-anesthetized animals were used to examine the
effect of Sar-[D-Phe8]des-Arg9-BK
(750 ng/kg i.a.) on MBF and MVR. Results are shown in figure 4. Overall changes of MABP and HR elicited by the
B1 agonist in halothane-anesthetized animals were similar
to those measured in barbiturate-anesthetized animals (compare data of
fig. 4 with those of fig. 3). Moreover patterns of MBF and MVR
decreases in halothane-anesthetized animals were nearly identical with
those of CBF and CVR decreases in barbiturate-anesthetized animals.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Sar-[D-Phe8]des-Arg9-BK, a
Lys-des-Arg9-BK analog that shares the high affinity of
this natural sequence, was shown previously to be resistant to
enzymatic breakdown by a variety of tissue enzymes from rabbits and to
elicit dose-dependent hypotensive effects when injected intraarterially
in LPS-treated rabbits (Drapeau et al., 1991). The
inhibition of
Sar-[D-Phe8]des-Arg9-BK-induced
hypotension, in the latter animal model, by sequence-related antagonist
of kinin B1 receptors, but not by HOE 140, a kinin B2 receptor, confirmed the participation of kinin
B1 receptors in this effect (Drapeau et al.,
1993). In addition to being a more potent hypotensive peptide than
des-Arg9-BK in LPS-treated animals,
Sar-[D-Phe8]des-Arg9-BK produced
longer lasting hypotensive episodes than the naturally occurring
B1 agonists, des-Arg9-BK and
Lys-des-Arg9-BK, in this animal model. The greater
metabolic stability of Sar-[D-Phe8]des-Arg9-BK, compared
with des-Arg9-BK and Lys-des-Arg9-BK, was
responsible for these differences (Drapeau et al., 1991). In
the present study we measured and compared the rate of elimination of
Sar-[D-Phe8]des-Arg9-BK and of
Lys-des-Arg9-BK from the plasma of anesthetized rabbits
which were not pretreated with LPS (hereafter called "normal"
animals). Our aim was to determine whether longer lasting hypotensive
episodes caused by
Sar-[D-Phe8]des-Arg9-BK in
LPS-treated animals, compared with Lys-des-Arg9-BK, might
rely upon differences of their rates of elimination from the plasma.
The choice of normal rather than LPS-treated animals was dictated by
the fact that high doses (3 mg/animal) of B1 agonists had
to be used to raise plasma concentrations to optically measurable
levels. Such high doses of B1 agonists were expected to
produce cardiovascular collapse in LPS-treated animals but were
essentially inert in intact animals (data not shown). The results
showed clearly that
Sar-[D-Phe8]des-Arg9-BK
disappears more slowly from plasma than Lys-des-Arg9-BK.
These results suggest that the longer duration of hypotensive effects
of Sar-[D-Phe8]des-Arg9-BK in
LPS-treated rabbits, compared with Lys-des-Arg9-BK or
des-Arg9-BK, may have a metabolic basis. However, this
conclusion is contingent upon the existence of identical metabolic
pathways in normal and LPS-treated animals.
Endogenous vasodilators such as prostacyclin and NO may participate in
the hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK in
LPS-treated rabbits, for both substances are released from isolated
rabbit arteries challenged with B1 agonists (see the introduction). In the present study we showed that indomethacin and
diclofenac do not reduce the amplitude of, but quicken the recoveries
from, hypotensive episodes caused by
Sar-[D-Phe8]des-Arg9-BK. These
results add further support to the hypothesis that PG release from
blood vessels may contribute to the prolonged hypotension caused by
Sar-[D-Phe8]des-Arg9-BK in
LPS-treated rabbits (Drapeau et al., 1991). However, the overall effect of PGs in this system is not a vasodilator one, as might
have been predicted from isolated vessels systems (see below). As far
as the participation of NO in
Sar-[D-Phe8]des-Arg9-BK-induced
hypotension is concerned, the results are negative (i.e., NO
is unlikely to be involved).
NG-nitro-L-arginine (NO synthase inhibitor) did
not alter the amplitude or duration of
Sar-[D-Phe8]des-Arg9-BK-induced
hypotension, despite the fact that this arginine derivative at the dose
utilized in this study was shown to inhibit the hypotensive effect of
ACH, a drug whose blood pressure-lowering effect in rabbits or rats
depends at least partially on NO release (Whittle et al.,
1989; Rees et al., 1990). Our conclusion that NO does not
contribute to the hypotensive action of
Sar-[D-Phe8]des-Arg9-BK in our
animal model is not in agreement with the results of Pruneau and
Belichard (1993) which indicate that the vasorelaxant effect of the
B1 agonist des-Arg9-BK in the isolated rabbit
carotid artery is mediated by endothelial NO release. However this
discrepancy is easily explained if one considers that: 1) the
participation of endothelial NO to the vasorelaxant (or contractile)
effects of B1 agonists in isolated rabbit arteries is
restricted to some blood vessels only (Bouthillier et al.,
1987; deBlois and Marceau, 1987; Ritter et al., 1989); and
2) any small contribution by NO to the hypotensive effect of
B1 agonists in LPS-treated rabbits is doomed to be masked
by presumably more prominent direct and indirect (i.e.,
PG-mediated) components of hypotensive effects of B1
agonists in this animal model.
Hypotensive responses to Sar-[D-Phe8]des-Arg9-BK in LPS-treated rabbits were not inhibited by animal treatment with dazmegrel, a thromboxane synthase inhibitor, MK-886, a leukotriene biosynthesis inhibitor, or by BN-50739, a PAF receptor antagonist. These results suggest that endogenous thromboxanes, leukotrienes and PAF (or PAF receptors), are unlikely to mediate the B1 agonist-induced hypotension in LPS-treated rabbits. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, atropine, a nonselective antagonist of muscarinic receptors, and propranolol, a nonselective beta adrenoceptor blocker, did not affect the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK in LPS-treated rabbits. These results indicate that Sar-[D-Phe8]des-Arg9-BK-induced hypotension is not caused by activation of vascular ATP-sensitive K+ channels, by the release of ACH or adrenaline from endogenous stores or by direct activation of vascular muscarinic or beta adrenergic receptors.
The hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK in our
animal model was characterized by prolonged decreases of MABP,
bradycardia, persistent decreases of CBF, FBF, MBF and CVP, and by
large decreases in CVR, FVR and MVR, which subsided within 30 to 0 sec
after injection of the B1 agonist and were followed in some
cases (CVR, FVR) by rebound increases, despite persisting hypotension.
No change of HTC was noted after injection of the B1
agonist. These results may be interpreted in the following way. First,
the absence of a change in HTC indicates that the hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK is not
the result of a loss of plasma volume caused by plasma extravasation.
Second, the acute decreases in CVR, FVR and MVR occurring within
seconds after injection of the B1 agonist, even though
transient, suggest that peripheral vasodilation and a decrease of TPR,
secondary to the direct effect of the B1 agonist on the blood vessels, may be the primary events responsible for the early decline of MABP. Third, the persistence of the hypotensive episode caused by
Sar-[D-Phe8]des-Arg9-BK, despite
the early recovery and/or rebound increases of vascular resistance in
some vascular beds, suggests that a reduction of CO may contribute to
the maintenance of the hypotension caused by the B1 agonist
in this animal model. Such interpretation is supported by our
observations that initial MABP decreases caused by
Sar-[D-Phe8]des-Arg9-BK were
associated with decreases of TPR but no significant changes of CO,
whereas the late phase of the hypotensive episode caused by the
B1 agonist was characterized mainly by a decrease of CO. The reduced CO may be caused by inappropriate compensatory responses (i.e., bradycardia instead of reflex tachycardia, excessive
increases of CVR and FVR) to the early acute decrease of MABP. A
decrease in cardiac contractility rather than in preload is more
likely, at least theoretically, to be responsible for the decrease in CO caused by the B1 agonist, because the CVP (an index of
preload) was back to base line at the time the CO was decreased. A
detrimental cardiac effect was not expected from a previous study of
the Langerdoff preparation based on hearts removed from LPS-treated
rabbits where a coronarovasodilator effect mediated by B1
receptors for kinins was measured (Regoli et al., 1981). The
absence of reflex tachycardia during the hypotensive episode caused by
Sar-[D-Phe8]des-Arg9-BK is a
relatively surprising event, because increases of HR and CO are the
usual compensatory responses to severe arterial hypotension (Rushmer,
1976). This unexpected event may point to a dysfunction of
baroreceptor-mediated cardiovascular reflexes in our animal model.
Both the bradycardia and rebound increases of CVR caused by
Sar-[D-Phe8]des-Arg9-BK were
attenuated in diclofenac-treated animals compared with control. These
results suggest that these two processes may be partially PG dependent.
Additional mechanisms besides PG release presumably are involved in the
HR-decreasing effect of the B1 agonist, because no reflex
tachycardia was observed even in diclofenac-treated animals during the
hypotensive effect of
Sar-[D-Phe8]des-Arg9-BK. The
increases of CVR occurring during the hypotensive episode caused by the
B1 agonist were converted into prolonged CVR decreases by
animal pretreatment with hexamethonium. These results suggest that
sympathetic neurons innervating blood vessels may also be involved in
such phenomena. The direct and/or indirect (i.e., via PG) activation of peripheral nociceptors functionally
coupled to vascular sympathetic neurons is perhaps one of the most
likely mechanisms by which
Sar-[D-Phe8]des-Arg9-BK may
elicit an increase of CVR (and presumably of MVR and FVR) in our animal
model. Evidence that B1 agonists may activate peripheral nociceptors via a PG-dependent mechanism was presented
previously (Davis and Perkins, 1994b; Walker et al., 1994).
The hypotensive episode elicited by Sar-[D-Phe8]des-Arg9-BK in LPS-treated, halothane-anesthetized animals was associated with bradycardia, persistent decreases of MBF and transient decreases of MVR. Prior increases of MABP and of MVR with the sympathomimetic vasopressor drug methoxamine produced little or no alteration of the ability of Sar-[D-Phe8]des-Arg9-BK to decrease MABP and HR levels in this animal model. However, the transient decreases of MVR and sustained decreases of MBF noted in methoxamine-free animals were converted into sustained decreases of MVR and sustained increases of MBF in methoxamine-treated animals. These results suggest that important changes of base-line (preinjection) MABP and/or arterial tone may modify both quantitatively and qualitatively the hemodynamic profile of B1 agonists. This conclusion is consistent with the results of DeWitt et al. (1994), which shows that the naturally occurring B1 agonist des-Arg9-BK elicits vasoconstriction under low arterial tone, but vasodilation under high arterial tone, in the pulmonary vascular bed of anesthetized cats.
In summary, i.a. injection of Sar-[D-Phe8]des-Arg9-BK in LPS-treated, anesthetized rabbits causes a prolonged episode of hypotension. Peripheral vasodilation is likely to be the primary event leading to the early decrease of MABP, whereas a reduction of CO may contribute to the maintenance of the hypotension induced by the B1 agonist in this animal model. Inappropriate compensatory responses to arterial hypotension, PG release and the slow rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma may be at the basis of the prolonged duration of the hypotensive episode elicited by the B1 agonist. These results provide additional circumstantial evidence that endogenous B1 agonists (e.g., des-Arg9-BK, Lys-des-Arg9-BK) may participate in the hemodynamic manifestations of septic shock.
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Acknowledgments |
|---|
We thank Elisabeth Lemay and Gaétane Rioux for typing this manuscript.
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Footnotes |
|---|
Accepted for publication September 4, 1996.
Received for publication February 6, 1996.
1 Supported in part by the Medical Research Council of Canada (Grant MT-12217) and the Quebec Heart and Stroke Foundation.
2 Recipient of a Studentship from Fonds pour la Formation de Chercheurs et l'Aide à la Recherche, Québec.
3 Scholar of the Fonds de la Recherche en Santé du Québec.
Send reprint requests to: Francis Rioux, Ph.D., Centre de recherche de l'Hôtel-Dieu de Québec, 11 Côte du Palais, Québec (Québec), Canada G1R 2J6.
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Abbreviations |
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ACH, acetylcholine; BK, bradykinin; CBF, carotid blood flow; CO, cardiac output; CVP, central venous pressure; CVR, carotid bed vascular resistance; DMSO, dimethyl sulfoxide; FBF, femoral blood flow; FVR, femoral bed vascular resistance; HR, heart rate; HPLC, high performance liquid chromatography; HTC, hematocrit; i.a., intraarterial; LPS, bacterial lipopolysaccharide; MABP, mean arterial blood pressure; MBF, mesenteric blood flow; mm Hg, millimeter of mercury; MVR, mesenteric bed vascular resistance; NO, nitric oxide; PAF, platelet-activating factor; PG, prostaglandin; Sar, sarcosine; T1/2, biological half-life in plasma; TPR, total peripheral resistance.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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in isolated smooth muscle cells: Correlation with B1 agonist-induced PGI2 production.
Br. J. Pharmacol.
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,
Sar-[D-Phe8]des-Arg9-BK; 
,
Lys-des-Arg9-BK; Cp, arterial plasma concentration).
*P < .05 and **P < .01, when compared with values of
animals given
Sar-[D-Phe8]des-Arg9-BK
(Student's t test).
). Data generated from the latter
two groups were compared with those obtained in diclofenac- and
C6-free (control) animals (
). Diclofenac and
C6 were given by i.a. injection 15 min before the
B1 agonist. 
