(Circulation. 1997;96:3570-3578.)
© 1997 American Heart Association, Inc.
Articles |
Cardiovascular Phenotype of a Mouse Strain With Disruption of Bradykinin B2-Receptor Gene
From Clinica Medica (P.M., N.G.) and Farmacologia (M.V.V., D.P., M.P.D., V.A.), University of Sassari (Italy); the National Institute of Biostructures and Biosystems (P.M.), Osilo, Italy; Farmacologia (C.E.), University of Ferrara (Italy); and Institute of Medicina Clinica (P.D.-F., R.S.), University of Ancona (Italy).
Correspondence to Paolo Madeddu, MD, Clinica Medica, University of Sassari, Viale S Pietro 8, 07100 Sassari, Italy. E-mail madeddu{at}ssmain.uniss.it
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Abstract |
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Background To evaluate the role of kinins in the regulation of cardiovascular function, we studied the phenotype of a mouse strain with disruption of the bradykinin B2-receptor gene (Bk 2r-/-).
Methods and Results Under basal conditions, tail-cuff blood pressure was higher in Bk2r-/- than in wild-type Bk2r+/+ and heterozygous Bk2r+/- mice (124±1 versus 109±1 and 111±2 mm Hg, respectively; P<.01 for both comparisons), a difference that was confirmed by measurements of intra-arterial blood pressure in unanesthetized mice. Heart weight was greater in Bk2r-/- than in Bk2r+/+ and Bk2r+/- mice (505±10 versus 449±12 and 477±10 mg/100 g body wt, P<.05). Chronic blockade of B2-receptors by Icatibant (50 nmol/100 g body wt twice a day SC) or inhibition of nitric oxide synthase by nitro-L-arginine-methyl ester (0.14 mmol/100 g body wt orally) increased the blood pressure of Bk2r+/+ to the levels of Bk2r-/- mice. Compared with the wild-type strain, both Bk2r-/- and Bk2r+/- mice showed exaggerated vasopressor responses to angiotensin II. In addition, chronic administration of an angiotensin AT1-receptor antagonist reduced the basal blood pressure of Bk2r-/- by 21±3 mm Hg (P<.05) to the levels of Bk2r+/+. No difference was detected between strains as far as plasma renin activity and the expression of renin and AT1-receptor genes are concerned. Chronic salt loading (0.84 mmol/g chow for 15 days) increased the blood pressure of Bk2r-/- and Bk2r+/- by 34±3 and 14±6 mm Hg, respectively, whereas it was ineffective in Bk2r+/+.
Conclusions Our results suggest that a normally functioning B2-receptor is essential for the maintenance of cardiovascular homeostasis in mice. Dysfunction of the kallikrein-kinin system could contribute to increase blood pressure levels by leaving the activity of vasoconstrictor agents unbalanced.
Key Words: kinins • bradykinin • receptors • genes
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Introduction |
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Kinins, derived from the enzymatic action of kallikrein on kininogen, are able to induce vasodilatation, diuresis, and natriuresis by promoting the release of endothelium-derived relaxing factors and prostaglandins.1 2 3 4 They act as local hormones by activating specific receptors named B1 and B2, most of the cardiovascular and renal effects being mediated by the B2-receptor.5 Chronic blockade of bradykinin (Bk) B2-receptors by D-Arg,(Hyp3, Thi5,D-Tic7,Oic8)-Bk (Icatibant) increases blood pressure (BP) in rats, provided that administration of the antagonist is combined with nonpressor doses of angiotensin II or deoxycorticosterone or it is started during early phases of postnatal life.6 7 8 These pharmacological studies suggest that the kallikrein-kinin system (KKS) could protect against the effects of vasoconstrictor agents. Therefore, a dysfunction of the KKS, leading to unbalanced predominance of vasoconstrictor and antinatriuretic systems, may contribute to the pathogenesis of arterial hypertension. Consistently, urinary kallikrein excretion was found to be decreased in essential hypertensive patients and in their normotensive offsprings9 as well as in various rat models of genetic hypertension.10 11 12 In addition, a restriction fragment length polymorphism of the kallikrein gene cosegregates with elevated BP levels in rats.13 As far as we know, no information is available regarding the possibility that polymorphisms of the B2-receptor gene influence BP phenotype. Unfortunately, linkage studies of complex diseases—such as hypertension—have limited power to detect genes of modest effect. A strategy that may overcome these limitations consists in phenotyping animals lacking a single candidate gene and then looking for gene polymorphisms in those hypertensive patients who share the mutant intermediate phenotype. To accomplish the first step of this strategy, we studied the cardiovascular phenotype of a mouse strain (Bk2r-/-) in which the B2-receptor gene has been targeting disrupted, thus leading to unresponsiveness to Bk in pharmacological preparations from ileum, uterus, and superior cervical ganglia.14 As a partial deficiency rather than complete absence of the receptor is more likely to occur in hypertensive patients, heterozygous animals were also evaluated. Studies were conducted under basal conditions and after the application of stresses such as (1) chronic alterations in salt intake, (2) chronic inhibition of nitric oxide synthase by nitro-L-arginine-methyl ester (L-NAME), and (3) acute or chronic administration of angiotensin II.
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Methods |
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Gene targeting was performed by transfecting embryonic stem cells derived from 129Sv/Ev mice with a vector designed to disrupt the entire coding sequence for the B2-receptor by homologous recombination.14 The targeting vector contains 1 kb of mouse genomic DNA immediately upstream of the coding sequence of the mouse B2-receptor, a neomycin resistance gene that replaces the coding sequence of the B2-receptor, 5.4 kb of mouse genomic DNA downstream of the B2-receptor coding sequence, and a tk gene. Injection of 129Sv/Ev embryonic cells, carrying the targeted mutation, into C57Bl/6J blastocysts produced highly chimerical mice. They were mated with 129Sv/Sl mice (a substrain closely related to 129Sv/Ev), and only the offspring (F1) that were heterozygous for the knock-out (thus having both sets of chromosomes of 129Sv origin) were used for subsequent mating to homozygousity (F2). Although not fully inbred, F2 Bk2r-/- mice are genetically very similar to parental substrains. This strategy has important advantages over the practice of breeding the chimerical animals to C57Bl/6J resulting in a hybrid 129SvxC57Bl/6J. Studies with F2 mice derived from these hybrids may suffer from the problem that the observed phenotype is influenced by genes linked to the mutation.15
The Bk2r-/- mice used in the present studies were provided by Merck Research Laboratories (Rahway, NJ). They were compared with 129Sv/J mice (Bk2r+/+, from Jackson Laboratory, Bar Harbor, Maine) and with heterozygous (Bk2r+/-) mice obtained by breeding pairs of Bk2r-/- and Bk2r+/+. The 129Sv/J substrain is closely related to the two substrains used to generate Bk2r-/- mice. However a limitation is represented by the fact that, although genetically similar, Bk2r+/+ and Bk2r-/- may differ at a limited number of loci others than the site of the mutation.
Mice were housed at a constant room temperature (24±1°C) and humidity (60±3%) with a 12-hour light/dark cycle. They had free access to chow (sodium, 0.12 mmol/g chow, Mucedola) and tap water, unless specified otherwise. All procedures complied with the standards for the care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Md) and were approved by the local Animal Care and Use Committee.
Blood Pressure and Heart Rate Measurements
Systolic BP and heart rate (HR) were measured by the
tail-cuff plethysmography method in unanesthetized mice
prewarmed for 10 minutes at 37°C in a thermostatically controlled
heating cabinet (50 cm wide, 50 cm high, and 80 cm long). Three
preliminary training sessions were performed over 1 week before the
experiment. With the mouse kept gently wrapped in a cotton hand towel,
the tail was passed through a miniaturized cuff (1 cm long, 0.8 cm id)
and a tail-cuff sensor (1.5 cm long, 0.3 or 0.4 cm id) that was
connected to an amplifier (Recorder 8002, Ugo Basile, Biological
Research Apparatus). The amplified pulse was recorded
during automatic inflation and deflation of the cuff. Tail-cuff
systolic BP is defined as the inflation pressure at which the
waveform becomes indistinguishable from baseline noise. As an inclusion
criterion, we required that at least 10 out of 12 measurements were
successful. Final BP value was obtained by averaging 10 to 12
successful readings. Experimental measurements were performed between
10 AM and 2 PM by a single investigator (M.V.V.
or D.P.) and then judged by an independent investigator in a blinded
fashion. HR was recorded automatically by a counter triggered by
the pulse wave.
To measure intra-arterial mean BP (MBP), a polyethylene catheter (PE-10, Clay Adams) was inserted into the left carotid artery and advanced into the thoracic aorta of mice anesthetized with 2,2,2-tribromoethanol (88 mmol/100 g body wt IP, Sigma-Aldrich). The catheter was tunneled under the skin and exteriorized at the back of the neck. The following day, MBP of unanesthetized, unrestrained mice was measured by connecting a Statham transducer (Gould) to the arterial catheter, and it was recorded on a Quartet recorder. Calibrations of instruments to measure tail-cuff or intra-arterial BP were performed with a mercury sphygmomanometer.
Cardiovascular Phenotype Under Basal
Conditions
Tail-cuff systolic BP and HR were measured on at least
three occasions over a 1-week period in male (Bk2r-/-,
n=50; Bk2r+/+, n=22; Bk2r+/-, n=12) and female
mice (Bk2r-/-, n=13; Bk2r+/+, n=10;
Bk2r+/-, n=12). MBP was measured on the following day.
Effects of Agonists and Antagonists of Bk-Receptors on
Direct and Tail-Cuff Blood Pressures
To confirm the absence of B2-receptors in the
knockout strain, the dose-MBP response curves to Bk (0.3 to 30
nmol/100 g body wt IV, Sigma-Aldrich) were assessed in
Bk2r-/-, Bk2r+/-, and Bk2r+/+
mice (n=8 per group), instrumented 1 day in advance with a carotid
catheter. For injection of the peptide, another PE-10 catheter was
inserted into the left jugular vein and exteriorized at the back of the
neck. Each dose was injected in a random order and sufficient time was
allowed (at least 30 minutes) for MBP to return to basal levels.
To confirm that the difference in basal BP between strains is due to the absence of the B2-receptor in knockout mice, we tested the BP effect of the B2-receptor antagonist Icatibant (50 nmol in 10 µL saline/100 g body wt twice a day SC), a generous gift from Hoechst Pharmaceutical Co (Frankfurt, Germany), in Bk2r-/- and Bk2r+/+ mice (n=7, each group). The dose and timing of administration were chosen on the basis of preliminary experiments in Bk2r+/+, showing that the vasodepressor effect of 1 nmol IV Bk is still inhibited by 70% at 12 hours after Icatibant, whereas the vasodepressor effects of acethylcholine and prostaglandin E2 are unaffected. Tail-cuff BP was measured in basal conditions, every 5 days during the administration of Icatibant and for additional 10 days after its discontinuation.
To evaluate whether the absence of the B2-receptor is associated with alterations in other components of the KKS, kinin levels were measured in urine obtained from mice on a normal sodium diet (n=6, each group). Urine samples were collected in ethanol (1:9 vol:vol), and sediment was discarded after centrifugation at 1500g for 10 minutes. Kinin determination was performed by radioimmunoassay according to Shimamoto et al.16 The sensitivity of the assay is 55 fmol/mL.
The BP responses of Bk2r-/- or Bk2r+/+ (n=5, each group) to activation or blockade of the B1-receptor were assessed by injection of the B1-receptor agonist Sar[D-Phe8]des-Arg9-Bk or the B1-receptor antagonist AcLys[D-ßNal7,Ile8]des-Arg9-Bk (both at 3 nmol/100 g body wt IV),17 generous gifts from Prof Domenico Regoli (University of Sherbrooke, Canada). MBP was recorded in basal conditions and for 10 minutes after injection.
Blood Pressure Effects of Alterations in Sodium Intake
Basal measurements were performed in Bk2r-/-,
Bk2r+/- and Bk2r+/+ mice on normosodic intake
(0.12 mmol sodium/g chow). Animals then were randomly assigned to
a normal (0.12 mmol/g chow, n=8 each group) or a high
(0.84 mmol/g chow, n=8 each group) sodium diet for the
following 15 days. Two additional groups of Bk2r-/- and
Bk2r+/+ mice received a low sodium diet (0.02
mmol/g chow, n=8 each group) for the same period of time. Other
nutrients remained unchanged. Tail-cuff BP, HR, and body weight were
measured every 5 days during the experimental period.
In the groups given chronic sodium load, 24-hour urine collections were obtained under basal conditions and then every day during the high sodium diet. Mice were housed in individual metabolic cages, which allowed for a high degree of accuracy in the measurement of food and water intake by the inclusion of spill catches. Urine volume (UV) was determined gravimetrically. Sodium concentration was measured by flame photometry.
At the end of the experiment, MBP was measured for 20 minutes in unanesthetized mice. The animals then were given an overdose of anesthesia, and the heart and both kidneys were removed, cleaned, washed in saline, blotted, and weighed.
Blood Pressure Effects of Angiotensin II and
AT1-Receptor Antagonist A-81988
The MBP response to acute angiotensin II injection
(6 to 150 pmol/100 g body wt IV, Sigma-Aldrich) was
evaluated in unanesthetized Bk2r-/- (n=10),
Bk2r+/- (n=8) and Bk2r+/+ (n=7). Each dose was
administered in a random order, and sufficient time was allowed (at
least 30 minutes) for MBP to return to basal levels.
The tail-cuff BP response to chronic angiotensin II infusion (15 nmol/100 g body wt per day IP, for 2 weeks) was evaluated in Bk2r-/-, Bk2r+/- and Bk2r+/+ (n=6, each group). Infusion was performed by using Alzet osmotic pumps (Alza Co) implanted into the abdominal cavity. Tail-cuff BP was measured twice in basal conditions, every 3 days during infusion and on two occasions after its discontinuation.
In an additional series of experiments, the chronic BP effect of A-81988 (2-{N-propyl-N-[(2'[1H-tetrazol-5-yl]biphenyl-4-yl)methyl]amino}pyridine-3-carboxylic acid), a nonpeptidic antagonist of angiotensin AT1-receptors,18 was evaluated in Bk2r-/- and Bk2r+/+. Animals were randomly assigned to a normal (0.12 mmol/g chow, n=8 each group), a low (0.02 mmol/g chow, n=8 each group), or a high (0.84 mmol/g chow, n=8 each group) sodium diet. After 15 days, they received the antagonist A-81988 (a generous gift from Abbott Laboratories, Abbott Park, Ill) for 10 days at the dose of 170 µg/100 g body wt per day orally. In the rat, the antagonist potency of A-81988 on the vasopressor response to intravenous angiotensin II is greater than that of losartan by a factor of at least 10. A-81988 has no affinity for adrenergic, cholinergic, endothelin, or PAF receptors, and it is >1000-fold more selective for AT1- versus AT2-receptors. In preliminary experiments, A-81988 (at the dose indicated above) was able to antagonize the vasopressor effect of 10 pmol intravenous angiotensin II by 75%. Tail-cuff BP was measured twice under basal conditions, every 5 days during A-81988 administration, and then after discontinuation of the compound.
Plasma Renin Activity of Bk2r-/- and
Bk2r+/+ Mice
For determination of plasma renin activity (PRA), mice (n=6 each
group) were anesthetized with 2,2,2-tribromoethanol, and blood
was collected from the orbital sinus into tubes containing
K3-EDTA. Plasma was stored at -40°C until assay.
Measurement of PRA was carried out by coated-tube radioimmunoassay
(RENCTK, Sorin Biomedica, Saluggia, Italy).
Renin and AT1-Receptor mRNA Levels in the Kidneys and
Hearts of Bk2r-/- and Bk2r+/+ Mice
Mice (n=6 each group) were killed by cervical dislocation.
Kidneys and hearts were removed and quickly frozen in liquid nitrogen.
Total RNA was extracted by ultracentrifugation through
cesium chloride gradient after sample
homogenization in guanidine thiocyanate. Northern
analysis with 10 µg of total RNA in each lane of duplicate
blots was performed with human renin and AT1 receptor cDNA
probes with conserved sequences for the two mouse renin genes and for
AT1A and AT1B genes.19 Each blot
was then rehybridized with mouse ß-actin cDNA. Densitometric
analysis of autoradiographic films was performed
with the Kodak Digital Science System coupled with BioMax 1D image
analysis software. The ratios of total renin on AT1
mRNA to ß-actin densitometric intensities were then calculated.
Blood Pressure Effects of Long-term L-NAME Administration
To test the effect of chronic blockade of nitric oxide synthase,
Bk2r-/- and Bk2r+/+ mice (n=8 per group) were
given L-NAME (0.14 mmol/100 g body wt for 20 days
orally). Tail-cuff BP and HR were measured twice under basal
conditions, every 5 days during L-NAME administration, and on two
occasions after its discontinuation.
Statistical Analysis
All data are expressed as mean±SEM.
Multivariate repeated-measures ANOVA was performed to
test for interaction between time and grouping factor.
Univariate ANOVA then was used among groups and over time.
Differences within and between groups were determined using paired or
unpaired Student's t test, respectively, with the
Bonferroni multiple-comparison adjustment.
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Results |
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Cardiovascular Phenotype Under Basal Conditions
As shown in Fig 1A
, Bk2r-/- mice showed higher tail-cuff BP values compared
with Bk2r+/+ and Bk2r+/- (males, 124±1 versus
109±1 and 111±2 mm Hg, respectively; females, 119±3 versus
106±2 and 112±2 mm Hg, respectively, P<.01 for both
comparisons). Intraindividual tail-cuff BP variability was similar
among groups (4.3±0.4 versus 6.4±0.7 and 5.9±0.7%, respectively
P=NS). Measurement of MBP in unanesthetized male mice confirmed
a difference among groups (144±3 versus 123±2 and 121±6 mm Hg,
P<.01 for both comparisons). A significant correlation was found
between direct and tail-cuff measurements (r=.925, P<.01).
The systematic observation of lower BP values with tail-cuff
plethysmography could be explained by the fact that animals were
prewarmed for better detection of tail artery pulse.
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As shown in Fig 1B
, HR values of male Bk2r-/- and
Bk2r+/- were higher compared with Bk2r+/+
(465±7 and 471±14 versus 424±8 bpm, P<.05), whereas in females no
group difference was observed. Intraindividual HR variability
coefficient was similar among groups (4.4±0.4%, 6.4±0.7%, and
5.7±0.8%, respectively, P=NS).
Effects of Agonists and Antagonists of Bk-Receptors on
Direct and Tail-Cuff Blood Pressures
As shown in Fig 2, Bk failed to
induce a vasodepressor response in Bk2r-/- mice, whereas
it induced similar dose-related MBP decreases in Bk2r-/-
and Bk2r+/-.
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MBP) induced by
graded doses of bradykinin (from 0.3 to 30 nmol/100 g body wt IV) in
bradykinin (Bk)2r-/- (filled circles),
Bk2r+/+ (open triangles), and Bk2r+/- (open
circles). Each group consisted of 8 mice. Values are mean±SEM. *P<.05
vs Bk2r+/+, §P<.05 vs Bk2r+/- mice.
As shown in Fig 3, chronic administration
of Icatibant did not alter the tail-cuff BP of Bk2r-/-.
By contrast, Icatibant increased the BP of Bk2r+/+ (from
107±2 to 121±2 mm Hg at 15 days, P<.05) to reach levels
similar to those of Bk2r-/-. Tail-cuff BP decreased
during the last days of Icatibant administration in
Bk2r+/+, and it was brought back to pretreatment levels
after antagonist discontinuation.
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Acute injection of Sar[D-Phe8]des-Arg9-Bk, a specific B1-receptor agonist, decreased the MBP of Bk2r-/- by 12±1 mm Hg (from 128±2 to 116±2 mm Hg at 4 minutes, P<.05), whereas it was ineffective in Bk2r+/+ mice (from 106±2 to 102±2 mm Hg, P=NS).
Acute injection of the B1-receptor antagonist AcLys[D-ßNal7,Ile8]des-Arg9-Bk exerted a greater vasopressor effect in Bk2r-/- (19±2 versus 6±1 mm Hg in Bk2r+/+, P<.01).
No difference was detected regarding urinary kinin excretion between Bk2r-/- and Bk2r+/+ mice (7.51±2.12 versus 8.57±2.90 pmol/24 hours, respectively, P=NS).
Blood Pressure Effects of Alterations in Salt Intake
As shown in Fig 4A, no change over
time was observed regarding tail-cuff BP in mice maintained on normal
sodium diet. Similarly, no time effect was detected as far as HR is
concerned. Body weight gain was similar among groups.
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As shown in Fig 4B, chronic salt loading increased the tail-cuff BP of
Bk2r-/- by 34±3 mm Hg (P<.01). This number was
significantly greater than that observed in Bk2r+/-
(14±6 mm Hg, P<.05). No significant change was detected in
Bk2r+/+ (6±4 mm Hg, P=NS). At the end of the
experiment, direct measurement revealed higher MBP levels in
Bk2r-/- (156±3 versus 131±6 and 119±2 mm Hg in
Bk2r+/- and Bk2r+/+, respectively, P<.05 for
both comparisons). In Bk2r-/-, the enhanced BP
sensitivity to salt was associated to a tachycardic response (from
500±12 to 597±16 bpm, P<.01), whereas HR did not change in
Bk2r+/- (from 486±17 to 491±8 bpm, P=NS) or
Bk2r+/+ (from 440±11 to 415±19 bpm, P=NS). During high
salt, body weight decreased in Bk2r-/- (from 23.5±0.9 to
21.7±1.1 g, P<.05), whereas it remained unchanged in
Bk2r+/- (from 21.7±0.7 to 22.1±0.5 g, P=NS) and
Bk2r+/+ (from 22.6±0.5 to 23.8±0.5 g, P=NS). Urinary
sodium excretion did not differ among groups under basal conditions
(Bk2r-/-, 0.55±0.02; Bk2r+/-, 0.56±0.03;
and Bk2r+/+, 0.57±0.02 mmol/d, P=NS). No group
difference was detected regarding cumulative sodium and water intake as
well as UV values during loading (data not shown). Cumulative urinary
sodium excretion was also similar among groups (Bk2r-/-,
57.5±4.8; Bk2r+/-, 58.2±4.0; and Bk2r+/+,
60.3±3.8 mmol in 15 days, P=NS). Thus the same sodium load was
excreted at the cost of increased BP levels in Bk2r-/-
and Bk2r+/-. A shift in the relation between natriuresis
and pressure in these two groups is indicated by the lower urinary
sodium to BP ratio during salt loading compared with wild-type mice
(Bk2r-/-, 0.37±0.02; Bk2r+/-, 0.44±0.03;
and Bk2r+/+, 0.51±0.02 mmol/mm Hg, P<.05 for
both comparisons).
As shown in Fig 4C, low sodium intake did not alter the tail-cuff BP of
Bk2r+/+ mice, whereas it increased that of
Bk2r-/- by 13±2 mm Hg (from 128±4 to 141±7
mm Hg, P<.05). No treatment effect was detected in HR within each
group. Body weight gain was similar among groups.
As shown in Table 1,
Bk2r-/- showed greater heart weight values compared with
Bk2r+/+ under normosodic conditions (505±12 versus 449±12
mg/100 g body wt, P<.05), whereas the heart weight of
Bk2r+/- was at intermediate levels (477±10 mg/100
g body wt). Similar differences among groups were detected under
hypersodic or hyposodic conditions. Kidney weight did not differ among
groups.
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Blood Pressure Effects of Angiotensin II and
AT1-Receptor Antagonist A-81988
As shown in Fig 5A, the curve
representing the MBP responses to different doses of
intravenous angiotensin II was shifted to the
left in Bk2r-/- and Bk2r+/- compared with
Bk2r+/+, whereas the slope was similar among groups.
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As shown in Fig 5B, chronic infusion of angiotensin II did
not alter the tail-cuff BP of Bk2r+/+, whereas it induced a
sustained BP increase in Bk2r-/- and Bk2r+/-
(31±7 and 29±6 mm Hg at 9 days, respectively). After
angiotensin II discontinuation, the BP of
Bk2r-/- and Bk2r+/- progressively returned
to baseline levels.
As shown in Fig 6, under normosodic
conditions, administration of A-81988 reduced the tail-cuff BP of
Bk2r-/- by 21 mm Hg, whereas it did not alter that
of Bk2r+/+. After discontinuation of A-81988, the BP of
Bk2r-/- returned to baseline levels. Similar BP effects
were observed under hyposodic conditions (Bk2r-/-, from
141±7 to 120±3 mm Hg, P<.05; Bk2r+/+, 112±3 to
103±5 mm Hg, P=NS). Under hypersodic conditions, A-81988 did not
alter the BP of Bk2r-/- or Bk2r+/+ (data not
shown).
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Renin and AT1-Receptor mRNA Levels in the Kidneys and
Hearts of Bk2r-/- and Bk2r+/+ Mice
No significant difference was detected between controls and
Bk2r-/- mice regarding renin and AT1-receptor
mRNA levels in renal tissue (Fig 7).
Similar results were obtained in cardiac tissues from the same animals
(data not shown).
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Plasma renin activity did not differ between Bk2r-/- and Bk2r+/+ mice (3.27±0.74 versus 3.38±0.58 ng/mL per hour, P=NS).
Blood Pressure Effects of L-NAME
As shown in Fig 8, basal tail-cuff
BP was higher in Bk2r-/- compared with
Bk2r+/+ mice. L-NAME increased BP in both strains, but the
magnitude of the effect was greater in Bk2r+/+ (45±5
versus 17±1 mm Hg in Bk2r-/- at 15 days, P<.05),
so that the initial difference between groups was nullified. After
discontinuation of L-NAME, BP returned to baseline values in both
strains. A bradycardic response was observed in Bk2r+/+,
whereas HR did not change in Bk2r-/- (-59±5 versus
-13±5 bpm, respectively, P<.05).
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A summary of Bk2r-/- and Bk2r+/-
cardiovascular phenotype under basal or
stimulated conditions is shown in Table 2.
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Discussion |
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We found that disruption of the Bk B2-receptor gene leads to increased BP levels. Under normosodic conditions, tail-cuff BP and intra-arterial MBP of knockout mice exceeded that of Bk2r+/+ by 15 and 21 mm Hg, respectively. The differences reported above are consistent with the BP increase observed in rats with early postnatal and lifelong blockade of the B2-receptor by Icatibant6 and with the predicted contribution of the kallikrein gene on overall BP variability.9 In addition, similar mildly elevated BP levels were observed in a rat strain that has been inbred for low urinary kallikrein excretion.20
The introduction of a transgene encoding for human Bk B2-receptor into the mouse genome reportedly generated a new strain in which overexpression of the receptor was associated with decreased BP levels.21 To test the hypothesis that mouse BP levels are inversely related to the expression of the B2-receptor gene, we determined the cardiovascular phenotype of heterozygous offspring derived from Bk2r-/- and Bk2r+/+ mice. Heterozygous animals are useful not only because they help understand the effect of varying the number of functional copies of the targeted gene but also because they resemble more closely a condition of "partial deficiency" that might occur in human hypertensive patients. The finding that Bk2r+/- are indistinguishable from wild-type animals regarding BP levels under normosodic conditions suggests that a partial defect in the B2-receptor may not suffice to alter mouse BP phenotype under basal conditions.
As expected, the vasodepressor response to Bk was completely absent in Bk2r-/-. The finding that Bk-induced hypotension was similar in Bk2r+/- and Bk2r+/+ is compatible with a great deal of redundancy or spare capacity of B2-receptors (ie, only a fraction has to be occupied to elicit a full response). In these conditions, a change in receptor expression is hard to detect by examining functional responses, such as vascular relaxation or uterine contraction.
We found that heart weight is increased in Bk2r-/-, possibly as a consequence of an augmented pressure load to the heart. However, we cannot exclude other possibilities including unopposed action of vasoconstrictors (eg, angiotensin II) or direct effect of receptor gene disruption on cardiomyocytes. In this context, it is important to note that increased heart weight values were also observed in heterozygous animals despite the fact that their BP did not differ from that of wild-type controls. Recent reports have suggested a direct inhibitory action of kinins on myocardial and vascular proliferation, instrumental for the cardiovascular protection exerted by ACE inhibitors.22 23 In myocardial ischemia, however, Bk facilitates sympathetic neurotransmission and norepinephrine release in the heart. Bk-induced norepinephrine release could be detrimental to the heart by causing vasoconstriction and arrhythmias.24 25 Further studies on transgenic mice would help to clarify the advantages and disadvantages of increasing kinin levels in the heart.
The most informative experiment to confirm that the altered BP phenotype of Bk2r-/- depends on receptor gene knock-out would be represented by returning the missing functional protein via direct systemic administration. Unfortunately, given the particularity of the protein under study (a receptor), these rescue experiments are not feasible. Therefore, we applied a different approach that consisted in evaluating the BP effect of a selective B2-receptor antagonist in wild-type and knockout mice. The finding that Icatibant increases the BP of Bk2r+/+ to the levels of Bk2r-/- favors the possibility that the BP difference between strains is due to the absence of a functional B2-receptor in mutant mice. Consistently, Icatibant reportedly normalizes the reduced BP of transgenic mice expressing the human Bk B2-receptor gene.21
Since targeted disruption of the B2-receptor gene might have altered other components of the KKS, experiments were performed to elucidate whether the final cardiovascular phenotype depends on unexpected effects of Bk not mediated by the B2-receptor. We found that urinary kinin levels are not altered in knock-out mice compared with wild-type controls. On the other hand, the vasodepressor response induced by a specific B1-receptor agonist was enhanced in Bk2r-/- and also augmented was the vasopressor effect exerted by a newly synthesized antagonist having high affinity and full selectivity for the B1-receptor. This implies that the B1-receptor is expressed in Bk2r-/-. We speculate that activation of the B1-receptor by endogenous kinins may compensate, at least in part, for the hemodynamic alterations induced by B2-receptor gene disruption.
Recently, Alfie et al26 reported that in Bk2r-/- mice the dose-BP response curve to acetylcholine, an endothelium-dependent vasodilator, is shifted to the right. To obtain further insight regarding the effect of mutation on endothelium-mediated vasodilatation, we tested the BP effects of nitric oxide synthase inhibition by chronic L-NAME administration. The finding that the magnitude of the vasopressor response to L-NAME was less in Bk2r-/- compared with wild-type mice suggests that the altered mutant phenotype could be simply the result of blunted basal level of activation of nitric oxide pathway by endogenous Bk, leading to amplification of vasoconstrictor activity. Attention was then focused on angiotensin II, since experimental evidence favors a functional interaction between the KKS and renin-angiotensin system.4 27 The slow vasopressor response to angiotensin II is reportedly enhanced in rats with chronic B2-receptor blockade as well as in kininogen-deficient rats, which congenitally lack the ability to generate kinins.7 28 Consistently, the responses to acute or chronic angiotensin II were found to be exaggerated in Bk2r-/- mice and even heterozygousity for the mutation was sufficient to elicit increased BP-sensitivity to the vasoconstrictor. By contrast, Alfie et al26 failed to demonstrate alterations in the acute BP response to angiotensin II. An important difference between the two studies is that Alfie and colleagues experiments were performed in mice under the effect of thiobutabarbital anesthesia, a factor that could have influenced the response to the vasoconstrictor agent, whereas ours were conducted in unanesthetized animals. In addition, the chronic effects of angiotensin II were not evaluated in the Alfie study.
A-81988, a potent and specific nonpeptidic AT1-receptor blocker, decreased the BP of Bk2r-/- either in normosodic conditions or after sodium deprivation, whereas in Bk2r+/+ it produced a modest decrease under hyposodic conditions only. The antagonist was ineffective in lowering BP in mice fed a hypersodic diet. These findings reinforce the view that kinins represent a mechanism capable of buffering the vascular action of endogenous angiotensin II. Analysis of PRA and of the expression of renin and AT1 receptor genes suggests that the mutant BP phenotype is the consequence of unbalanced rather than enhanced action of the renin-angiotensin system. This interpretation is consistent with the observation that also the gene expression of the other components (eg, angiotensinogen and angiotensin-converting enzyme) is not altered in mutant mice (Paolo Madeddu, unpublished observations, 1997).
In a previous study, Alfie et al26 reported that BP sensitivity to salt is exaggerated in Bk2r-/- mice compared with Bk2r+/+. The sodium load used by Alfie et al was extremely high, since mice passed from a normal diet containing 0.2% sodium in food to a high sodium diet containing 3.15% in food, plus 1% saline in drinking water. In preliminary studies, we found that such an increase in sodium intake causes profound retardation in growth and occasionally death. Therefore we decided to evaluate the BP effect of a milder sodium load. To this aim, sodium content was increased in the food from 0.25% to 1.75%, whereas no salt was added to drinking water. Even in this more "physiological" setting, Bk2r-/- showed an exaggerated BP sensitivity compared with wild-type mice. In Bk2r-/- mice, the BP response to salt loading was similar to that found in other species with functional impairment of the KKS such as rat strains inbred for low urinary kallikrein or for kininogen deficiency, Wistar rats with early postnatal blockade of the Bk B2-receptor, and essential hypertensive patients with depressed urinary kallikrein.6 20 29 30 Interestingly, heterozygous mice also showed an augmented BP response to high sodium (though less pronounced than Bk2r-/-), whereas under normosodic conditions they did not differ from wild-type animals. A gene-dosage effect on BP phenotype under hypersodic conditions could be attributable to the influence of salt loading on endogenous kinin concentration, kinin receptor expression, or kinetics of agonist-receptor interaction.31 Metabolic studies indicate that urinary excretion of salt is normal in Bk2r-/- and Bk2r+/- mice, a condition that is made possible by a shift in the BP-natriuresis relation. Despite our efforts to reduce the unwanted effects of high salt diet on growth, homozygous mutant mice significantly lost weight compared with the other groups. No group difference was detected in water and food intake or in urinary volume. However, neither water content of feces nor total body fluid volume was measured. Thus we cannot exclude that homozygous mutants became dehydrated on high salt. Another possibility is that the energy consumption was increased in these animals.
A mild BP increase was observed in Bk2r-/- under sodium deprivation. We speculate that sodium depletion could have amplified the vasoconstrictor influence of angiotensin II, since BP was normalized by blockade of the AT1-receptor.
The finding that Bk2r-/- mice have increased HR under basal conditions, with further tachycardic response to salt loading, was unexpected since Bk is able to enhance the release of norepinephrine via the B2-receptor.24 Interestingly, similar accelerated HR was observed in rats given Icatibant in utero,6 thus suggesting that peripheral and/or central B2-receptors may be involved in the early developmental processes that determine the adult HR phenotype in rodents. Reduced parasympathetic activity, sympathoexitation, and/or alteration in baroreceptor reflex sensitivity could be responsible for the alterations in HR. Consistent with the latter possibility is our previous observation that rat baroreceptor reflex response to increments in BP is reduced by central administration of Icatibant.32 However, the mechanisms mediating the abnormal HR responses of animals with genetic or pharmacological manipulation of the B2-receptors remain unknown.
In conclusion, our findings indicate that a normally functioning B2-receptor is essential for the maintenance of cardiovascular homeostasis and encourage the search for human functional variants of the B2-receptor gene. Recognition of such polymorphisms may help identify hypertensive patients likely to benefit from reduction of excessive salt intake.
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Acknowledgments |
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This work was supported in part by a grant from the Minister of Universities and Scientific Research. We wish to thank Prof Edwin Fink (Ludwig Maximilians University, Munich, Germany) for measurements of urinary kinins.
Received March 13, 1997; revision received June 13, 1997; accepted July 15, 1997.
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 P. Meneton, M. Bloch-Faure, A. A. Hagege, H. Ruetten, W. Huang, S. Bergaya, D. Ceiler, D. Gehring, I. Martins, G. Salmon, et al. Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice PNAS, February 15, 2001; (2001) 51619598. [Abstract] [Full Text]
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C. Emanueli, A. Minasi, A. Zacheo, J. Chao, L. Chao, M. B. Salis, S. Straino, M. G. Tozzi, R. Smith, L. Gaspa, et al. Local Delivery of Human Tissue Kallikrein Gene Accelerates Spontaneous Angiogenesis in Mouse Model of Hindlimb Ischemia Circulation, January 2, 2001; 103(1): 125 - 132. [Abstract] [Full Text] [PDF]
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N.-E. Rhaleb, X.-P. Yang, M. Nanba, E. G. Shesely, and O. A. Carretero Effect of Chronic Blockade of the Kallikrein-Kinin System on the Development of Hypertension in Rats Hypertension, January 1, 2001; 37(1): 121 - 128. [Abstract] [Full Text] [PDF]
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C. Emanueli, A. Zacheo, A. Minasi, J. Chao, L. Chao, M. B. Salis, T. Stacca, S. Straino, M. C. Capogrossi, and P. Madeddu Adenovirus-Mediated Human Tissue Kallikrein Gene Delivery Induces Angiogenesis in Normoperfused Skeletal Muscle Arterioscler Thromb Vasc Biol, November 1, 2000; 20(11): 2379 - 2385. [Abstract] [Full Text] [PDF]
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J. B. Pesquero, R. C. Araujo, P. A. Heppenstall, C. L. Stucky, J. A. Silva Jr., T. Walther, S. M. Oliveira, J. L. Pesquero, A. C. M. Paiva, J. B. Calixto, et al. Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors PNAS, June 14, 2000; (2000) 120035997. [Abstract] [Full Text]
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C. Emanueli, M. B. Salis, J. Chao, L. Chao, J. Agata, K.-F. Lin, A. Munao, S. Straino, A. Minasi, M. C. Capogrossi, et al. Adenovirus-Mediated Human Tissue Kallikrein Gene Delivery Inhibits Neointima Formation Induced by Interruption of Blood Flow in Mice Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1459 - 1466. [Abstract] [Full Text] [PDF]
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D. Wang, H. Yoshida, Q. Song, L. Chao, and J. Chao Enhanced renal function in bradykinin B2 receptor transgenic mice Am J Physiol Renal Physiol, March 1, 2000; 278(3): F484 - F491. [Abstract] [Full Text] [PDF]
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P. Madeddu, C. Emanueli, R. Maestri, M. B. Salis, A. Minasi, M. C. Capogrossi, and G. Olivetti Angiotensin II Type 1 Receptor Blockade Prevents Cardiac Remodeling in Bradykinin B2 Receptor Knockout Mice Hypertension, January 1, 2000; 35(1): 391 - 396. [Abstract] [Full Text] [PDF]
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L. J. Dell’Italia and S. Oparil Bradykinin in the Heart : Friend Or Foe? Circulation, December 7, 1999; 100(23): 2305 - 2307. [Full Text] [PDF]
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C. Emanueli, R. Maestri, D. Corradi, R. Marchione, A. Minasi, M. G. Tozzi, M. B. Salis, S. Straino, M. C. Capogrossi, G. Olivetti, et al. Dilated and Failing Cardiomyopathy in Bradykinin B2 Receptor Knockout Mice Circulation, December 7, 1999; 100(23): 2359 - 2365. [Abstract] [Full Text] [PDF]
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P. Madeddu, C. Emanueli, N.-E. Rhaleb, E. G. Shesely, and O. A. Carretero Can Knockout Mice Help Dissect Relevant Genes in Hypertension? Evidence and Confounding Factors • Response Hypertension, December 1, 1999; 34 (6): e14 - e15. [Full Text] [PDF]
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M. J. ZYCHMA, J. GUMPRECHT, E. ZUKOWSKA-SZCZECHOWSKA, and W. GRZESZCZAK Polymorphisms in the Genes Encoding for Human Kinin Receptors and the Risk of End-Stage Renal Failure: Results of Transmission/DisequilibriumTest J. Am. Soc. Nephrol., October 1, 1999; 10(10): 2120 - 2124. [Abstract] [Full Text]
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L. Cervenka, L. M. Harrison-Bernard, S. Dipp, G. Primrose, J. D. Imig, and S. S. El-Dahr Early Onset Salt-Sensitive Hypertension in Bradykinin B2 Receptor Null Mice Hypertension, August 1, 1999; 34(2): 176 - 180. [Abstract] [Full Text] [PDF]
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N. TAKAHASHI and O. SMITHIES Gene Targeting Approaches to Analyzing Hypertension J. Am. Soc. Nephrol., July 1, 1999; 10(7): 1598 - 1605. [Abstract] [Full Text]
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D. J. Campbell, A. Kladis, T. A. Briscoe, and J. Zhuo Type 2 Bradykinin-Receptor Antagonism Does Not Modify Kinin or Angiotensin Peptide Levels Hypertension, May 1, 1999; 33(5): 1233 - 1236. [Abstract] [Full Text] [PDF]
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P. Madeddu, M. B. Salis, A. F. Milia, C. Emanueli, R. Guerrini, D. Regoli, and G. Calo Cardiovascular Effects of Nociceptin in Unanesthetized Mice Hypertension, March 1, 1999; 33(3): 914 - 919. [Abstract] [Full Text] [PDF]
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 W. C. Wolf, R. A. Harley, D. Sluce, L. Chao, and J. Chao Localization and Expression of Tissue Kallikrein and Kallistatin in Human Blood Vessels J. Histochem. Cytochem., February 1, 1999; 47(2): 221 - 228. [Abstract] [Full Text]
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N.-E. Rhaleb, H. Peng, M. E. Alfie, E. G. Shesely, and O. A. Carretero Effect of ACE Inhibitor on DOCA-Salt– and Aortic Coarctation–Induced Hypertension in Mice : Do Kinin B2 Receptors Play a Role? Hypertension, January 1, 1999; 33(1): 329 - 334. [Abstract] [Full Text] [PDF]
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P. Madeddu, A. F. Milia, M. B. Salis, L. Gaspa, W. Gross, A. Lippoldt, and C. Emanueli Renovascular Hypertension in Bradykinin B2-Receptor Knockout Mice Hypertension, September 1, 1998; 32(3): 503 - 509. [Abstract] [Full Text] [PDF]
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C. Emanueli, E. Fink, A. F. Milia, M. B. Salis, M. Conti, M. P. Demontis, and P. Madeddu Enhanced Blood Pressure Sensitivity to Deoxycorticosterone in Mice With Disruption of Bradykinin B2 Receptor Gene Hypertension, June 1, 1998; 31(6): 1278 - 1283. [Abstract] [Full Text] [PDF]
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Z. Saifudeen, H. Du, S. Dipp, and S. S. El-Dahr The Bradykinin Type 2 Receptor Is a Target for p53-mediated Transcriptional Activation J. Biol. Chem., May 12, 2000; 275(20): 15557 - 15562. [Abstract] [Full Text] [PDF]
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P. Meneton, M. Bloch-Faure, A. A. Hagege, H. Ruetten, W. Huang, S. Bergaya, D. Ceiler, D. Gehring, I. Martins, G. Salmon, et al. Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice PNAS, February 27, 2001; 98(5): 2634 - 2639. [Abstract] [Full Text] [PDF]
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J. B. Pesquero, R. C. Araujo, P. A. Heppenstall, C. L. Stucky, J. A. Silva Jr., T. Walther, S. M. Oliveira, J. L. Pesquero, A. C. M. Paiva, J. B. Calixto, et al. Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors PNAS, July 5, 2000; 97(14): 8140 - 8145. [Abstract] [Full Text] [PDF]
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X.-P. Yang, Y.-H. Liu, D. Mehta, M. A. Cavasin, E. Shesely, J. Xu, F. Liu, and O. A. Carretero Diminished Cardioprotective Response to Inhibition of Angiotensin-Converting Enzyme and Angiotensin II Type 1 Receptor in B2 Kinin Receptor Gene Knockout Mice Circ. Res., May 25, 2001; 88(10): 1072 - 1079. [Abstract] [Full Text] [PDF]
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